Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries

ABSTRACT

The invention provides compositions and methods for treatment of tendon and ligament injuries and/or repair of damaged tendons and ligament. The invention provides compositions comprising a biocompatible matrix and platelet-derived growth factor (PDGF).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/556,555, filed Sep. 9, 2009, now U.S. Pat. No. 8,870,954, whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/191,641, filed Sep. 9, 2008, U.S. ProvisionalApplication No. 61/144,088, filed Jan. 12, 2009, and U.S. ProvisionalApplication No. 61/144,126, filed Jan. 12, 2009; this application isalso a continuation-in-part of PCT Application No. PCT/US2009/056418,filed on Sep. 9, 2009; the entireties of which are herein incorporatedby reference.

TECHNICAL FIELD

This invention relates to compositions and methods for treatment oftendon or ligament injuries, such as ruptured, severed, tom, ortransected tendons or ligaments or tendon or ligament detachment frombone, and in particular to methods for treatment of tendon or ligamentinjuries by administering compositions comprising a biocompatible matrixin combination with platelet-derived growth factor (PDGF).

BACKGROUND OF THE INVENTION

Tendons and ligaments are the tough fibers that connect muscle to boneor bone to bone, but tendons and ligaments can be ruptured, severed, ordetached from the bone for a variety of reasons. Such tendon or ligamentinjuries may generally occur due to or resulting from direct trauma tothe affected tendon/ligament, weakening of the tendon/ligament due toadvanced age, eccentric loading, repetitive motions, overuse and/orincreased stress or activity. Such acute injuries are quite dramatic andusually leave the individual unable to move the affected joint.

The most common areas of tendon rupture, tendon severance, or detachmentfrom the bone are (1) the quadriceps (a group of four muscles, thevastus lateralis, vastus medialis, vastus intermedius, and the rectusfemoris) which come together just above the kneecap (patella) to formthe patellar tendon; (2) the Achilles tendon, located on the back(posterior) portion of the foot just above the heel. The Achilles tendonserves as the attachment of the calf muscle (gastrocnemius muscle) tothe heel of the foot (the calcaneus bone); (3) the rotator cuff, locatedin the shoulder and composed of four muscles (the supraspinatus (themost common tendon ruptured), infraspinatus, teres minor, andsubscapularis); (4) the biceps of the arm, which functions as a flexorof the elbow. Ruptures of the biceps are classified into proximal(close) and distal (far) types; and (5) the flexor tendons of the hand,such as the flexor brevis and longus. The most common areas of ligamentrupture, ligament severance, or detachment from the bone are theanterior cruciate ligament (ACL), posterior cruciate ligamen (PCL), andmedial collateral ligament (MCL). For almost all tendon and ligamentinjuries there may be consideration pain (either acute or chronic), lossof motion and weakness of the affected joint or limb. For a ruptured ordetached tendon/ligament, surgery is the most common course oftreatment, in order to secure the tendon or ligament to its bone, or toreconnect the ruptured or severed ends of the affected tendon/ligament.For other tendon/ligament injuries, common treatments include rest, ice,NSAIDs, corticosteroid injections, heat, and ultrasound. However,despite decades of research and increasing clinical attention to theseinjuries, their clinical outcomes remain unpredictable.

With respect to the quadricepts, rupture of the patellar tendon is arelatively infrequent, yet disabling injury, which is most commonly seenin patients less than 40 years of age. It tends to occur during athleticactivities when a violent contraction of the quadriceps muscle group isresisted by the flexed knee. Rupture usually represents the final stageof a degenerative tendinopathy resulting from repetitive microtrauma tothe patellar tendon.

With respect to the Achilles tendon, both athletes and non-athletes areat risk for developing injuries at all ages, with most injuriesoccurring in men between the ages of 30 and 50 years of age (Boyden, E.,et al., Clin Orthop, 317:150-158 (1995); Hattrup, S. and Johnson, K,Foot and Ankle, 6: 34-38 (1985); Jozsa, L., et al., Acta OrthopScandinavica, 60:469-471(1989)). Achilles tendonitis and tendinosis arealso common in individuals whose work puts stress on their ankles andfeet, as well as in “weekend warriors,” those who are less conditionedand participate in athletics only on weekends or infrequently.

In the case of rotator cuff injuries, notwithstanding advances insurgical instrumentation and techniques, the current techniques fallshort of producing an enduring repair, with some studies citing failurerates as high as 94%. Failure of tendon repairs may be attributed topoor healing of the damaged tendon and poor reattachment of the injuredtendon to the bone.

A firm attachment of ligament to bone is also essential for manyligament reconstruction procedures. Successful ligament substitutionprocedures, such as anterior cruciate ligament reconstruction, requirefixation of a tendon graft into a bone tunnel and progressive ingrowthof bone into the tendon to create a biological attachment between thebone and the tendon. Histological and biomechanical studies show that itgenerally requires six to twelve weeks after the transplantation of atendon graft to a bone to achieve bone ingrowth, tendon-bone attachment,mineralization, and greater collagen-fiber continuity between the tendonand the bone. See, Rodeo S. A. et al., Tendon-Healing in a Bone Tunnel,75(12): 1795-1803 (1993).

Accordingly, there is a need to provide new compositions and new methodsof treatment for various tendon/ligament injuries and tendon/ligament tobone attachment to improve the healing response associated with surgicalrepairs or other non-surgical treatments.

All references cited herein, including, without limitation, patents,patent applications and scientific references, are hereby incorporatedby reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides for compositions and methods of treatmentof tendon or ligament injuries.

In one aspect of the invention, a composition is provided comprising abiocompatible matrix and platelet-derived growth factor (PDGF), whereinthe biocompatible matrix comprises pores, wherein the biocompatiblematrix has a porosity of at least about 80%, and wherein at least about50% of the PDGF is released within about 24 hours.

In another aspect, the invention provides a method for treatment of atendon injury or a ligament injury not involving a bone in an individualcomprising administering to an affected site of the injury of theindividual an effective amount of a composition comprising: abiocompatible matrix and platelet-derived growth factor (PDGF), whereinthe biocompatible matrix comprises pores, wherein the biocompatiblematrix has a porosity of at least about 80%, and wherein at least about50% of the PDGF is released within about 24 hours. In some embodiments,the method further comprises the step of mechanically stabilizing thetendon or ligament injury. In some embodiments, the step of stabilizingthe tendon or ligament injury comprises suturing the tendon or ligamentinjury, wherein the sutured tendon or ligament is positioned such thatthe ends of the injured tendon or ligament are substantiallyre-approximated. In some embodiments, the step of administeringcomprises administering to the affected site of the injury of saidindividual the effective amount of the composition using a syringe.

In another aspect, the invention provides a method for attaching orreattaching a tendon to a bone in an individual comprising administeringto the individual an effective amount of a composition comprising abiocompatible matrix and platelet-derived growth factor (PDGF) at aninterface between the tendon and the bone, wherein the biocompatiblematrix comprises pores, wherein the biocompatible matrix has a porosityof at least about 80%, and wherein at least about 50% of the PDGF isreleased within about 24 hours.

In another aspect, the invention provides a method for attaching orreattaching a ligament to a bone in an individual comprisingadministering to the individual an effective amount of a compositioncomprising a biocompatible matrix and platelet-derived growth factor(PDGF) at an interface between the ligament and the bone, wherein thebiocompatible matrix comprises pores, wherein the biocompatible matrixhas a porosity of at least about 80%, and wherein at least about 50% ofthe PDGF is released within about 24 hours.

In another aspect, the invention provides a kit for treatment of atendon or a ligament injury not involving a bone in an individualcomprising a first container comprising a biocompatible matrix and asecond container comprising a platelet-derived growth factor (PDGF)solution, wherein the biocompatible matrix comprises pores, wherein thebiocompatible matrix has a porosity of at least about 80%, and whereinat least about 50% of the PDGF is released within about 24 hours.

In yet another aspect, the invention provides a kit for attaching atendon or a ligament to a bone in an individual comprising a firstcontainer comprising a biocompatible matrix and a second containercomprising a platelet-derived growth factor (PDGF) solution, wherein thebiocompatible matrix comprises pores, wherein the biocompatible matrixhas a porosity of at least about 80%, and wherein at least about 50% ofthe PDGF is released within about 24 hours.

Any of the compositions as described herein may be used in any of themethods or kits as described herein. The various embodiments asdescribed below may be used in conjunction with any aspects of theinvention, as would be apparent to one of ordinary skill in the art.

In some embodiments, the biocompatible matrix has a porosity of at leastabout 25%, at least about 50%, at least about 75%, at least about 85%,at least about 90%, at least about 92%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, about 80% to about 95%, about 85% to about 95%,about 85% to about 90%, about 90% to about 92%, or about 92% to about95%.

In some embodiments, the pores have an average area ranging from about800 um² to about 3,000 um², about 13,000 um² to about 50,000 um², about2500 um² to about 20,000 um², about 4500 um² to about 20,000 um², about5000 um² to about 19,000 um², about 6000 um² to about 18,000 um², about6000 um² to about 15,000 um², or about 5000 um² to about 16000 um².

In some embodiments, the pores have an average perimeter ranging fromabout 100 μm to about 200 μm, about 400 μm to about 800 μm, about 200 μmto about 500 μm, about 200 μm to about 600 μm, about 300 μm to about 600μm, or about 300 μm to about 500 μm.

In some embodiments, the pores have average diameters ranging from about1 μm to about 1 mm. In some embodiments, the pores have averagediameters at least about 5 μm, at least about 10 μm, at least about 20μm, at least about 30 μm, at least about 40 μm, or at least about 50 μm,about 5 μm to about 500 μm, about 10 μm to about 500 μm, about 50 μm toabout 500 μm, about 100 μm to about 500 μm, or about 100 μm to about 300μm.

In some embodiments, the pores comprise interconnected pores.

In some embodiments, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, about 50% to about 95%, about 60% to about 95%, about 70% toabout 95%, about 80% to about 95%, about 90% to about 95%, about 50% toabout 85%, about 60% to about 85%, about 70% to about 85%, or about 50%to about 80% of the PDGF is released within about 24 hours. In someembodiments, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,about 50% to about 95%, about 60% to about 95%, about 70% to about 95%,about 80% to about 95%, about 90% to about 95%, about 50% to about 85%,about 60% to about 85%, about 70% to about 85%, or about 50% to about80% of the PDGF is released within about 1 hour, about 6 hours, about 8hours, about 12 hours, or about 48 hours. In some embodiments, the PDGFrelease is measured in vivo. In some embodiments, the PDGF release ismeasured in vitro. In some embodiments, the PDGF is released into thesurrounding region. In some embodiments, the PDGF is released onto aninjured tendon or ligament. In some embodiments, the PDGF is releasedonto the surface of the bone near the point of bone-tendon orbone-ligament attachment. In some embodiments, the PDGF is released intothe surrounding media.

In some embodiments, the biocompatible matrix is resorbed within about 1month, about 3 months, about 6 months, or about 9 months of in vivoadministration. In some embodiments, the biocompatible matrix isresorbed within about 30 days, about 25 days, about 21 days, about 18days, about 15 days, about 10-14 days, or about 10 days of in vivoadministration.

In some embodiments, the biocompatible matrix comprises collagen. Insome embodiments, the collagen comprises Type I collagen. In someembodiments, the collagen is cross-linked.

In some embodiments, the collagen is soluble. In some embodiments, thecollagen is insoluble. In some embodiments, the collagen comprises asoluble collagen monomer component and an insoluble collagen polymercomponent. In some embodiments, the ratio of soluble collagen monomersto insoluble collagen polymers is about 1:10, about 1:9, about 1:8,about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, orabout 1:1.

In some embodiments, the biocompatible matrix further comprises aglycosaminoglycan. In some embodiments, the glycosaminoglycan ischondroitin sulfate. In some embodiments, the glycosaminoglycan is notchondroitin sulfate.

In various embodiments, the composition is a gel, particle, powder,paste, sheet, pad, patch, or sponge. In some embodiments, thecomposition isflowable.

In some embodiments, the biocompatible matrix is COLLATAPE®. In someembodiments, the biocompatible matrix is BIOBLANKET®. In someembodiments, the biocompatible matrix is not BIOBLANKET®. In someembodiments, the biocompatible matrix does not oxidize PDGF.

In some embodiments, the PDGF is present as a solution comprising PDGF,wherein the concentration of PDGF in the solution is about 0.1 to about10.0 mg/ml, about 5 mg/ml to about 20 mg/ml, about 0.1 mg/ml to about2.0 mg/ml, about 0.1 mg/ml to about 0.4 mg/ml, or about 0.9 mg/ml toabout 1.5 mg/ml. In some embodiments, the PDGF solution is about 0.15mg/ml, about 0.3 mg/ml, about 1.0 mg/ml, about 1.5 mg/ml, about 2.0mg/ml, or about 10.0 mg/ml.

In some embodiments of the present invention, PDGF is a PDGF homodimer,in other embodiments, PDGF is a heterodimer, including for example,PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, and mixtures andderivatives thereof. In some embodiments, PDGF comprises PDGF-BB. Inother embodiments, PDGF comprises a recombinant human (rh) PDGF such asrecombinant human PDGF-BB (rhPDGF-BB).

In some embodiments of the present invention, PDGF is a PDGF fragment.In some embodiments, rhPDGF-B comprises the following fragments: aminoacid sequences 1-31, 1-32, 33-108, 33-109, and/or 1-108 of the entire Bchain.

In some embodiments, cells infiltrate the composition of the presentinvention within about 3 weeks, about 2 weeks, about 1 week, about 7days, about 6 days, about 5, about 4, about 3, about 2, or about 1day(s) after exposure to the composition. In some embodiments, the cellsare tenocytes. In some embodiments, the cells are osteoblasts. In someembodiments, the cells are ligament cells.

In some embodiments, compositions and methods of the present inventionare useful in the attachment of tendons to bone or ligaments to bone,and may be applied to any tendon or ligament attachment. In someembodiments, compositions and methods of the present invention enhancetendon attachment to bone or ligament attachment to bone bystrengthening the tendon and/or bone and ligament and/or bone at thesite of tendon/ligament attachment to the bone. Other types of damagedor injured tendons and/or ligaments, such as a tendon/ligamentexhibiting tearing, delamination, and/or any other strain ordeformation, may also be treated by the methods of the invention. Asingle type of injury may be treated, or more than one type of injurymay be treated simultaneously. In some embodiments, the treatmentinvolves treating a tendon. In some embodiments, the treatment involvestreating a ligament. In some embodiments, a tendon graft is used for thespecific treatment of tendon and/or ligament tissues. In otherembodiments, a ligament graft is used for the specific treatment oftendon and/or ligament tissues.

In some embodiments, compositions and methods of the present inventionare useful both in the attachment or reattachment of tendons to bone andany tendon injuries not involving a bone. In some embodiments,compositions and methods of the present invention are useful both inenhancing attachment or reattachment of tendons to bone and any tendoninjuries not involving a bone. In other embodiments, compositions andmethods of the present invention are useful both in the attachment orreattachment of ligaments to bone and any ligament injuries notinvolving a bone. In other embodiments, compositions and methods of thepresent invention are useful both in enhancing attachment orreattachment of ligaments to bone and any ligament injuries notinvolving a bone.

In some embodiments, the tendon which may be treated by compositions andmethods of the present invention include, but are not limited to,tendons of the subscapularis, supraspinatus, infraspinatus, teres minor,rectus femoris, tibialis posterior, quadriceps femoris, biceps brachii,as well as the Achilles Tendon, patellar tendon, abductor and adductortendons, or other tendons of the hip, the common extensor tendon, commonflexor tendon, flexor digitorum superficialis tendons, extensordigitorum and extensor minimi tendons, or other tendons of the arm andhand. In some embodiments, the tendon which may be treated bycomposition and methods of the present invention is selected from thegroup consisting of: patellar tendon, anterior tibialis tendon, Achillestendon, Hamstring tendon, semitendinosus tendon, gracilis tendon,abductor tendon, and adductor tendon. In some embodiments, the tendonwhich may be treated by compositions and methods of the presentinvention is selected from the group consisting of supraspinatus tendon,infraspinatus tendon, subscapularis tendon, teres minor tendon (rotatorcuff complex), flexor tendon, rectus femoris tendon, tibialis posteriortendon, and quadriceps femoris tendon. In some embodiments, tendon whichmay be treated by compositions and methods of the present invention isselected from the group consisting of patellar tendon, anterior tibialistendon, Achilles tendon, Hamstring tendon, semitendinosus tendon,gracilis tendon, abductor tendon, adductor tendon, supraspinatus tendon,infraspinatus tendon, subscapularis tendon, teres minor tendon (rotatorcuff complex), flexor tendon, rectus femoris tendon, tibialis posteriortendon, and quadriceps femoris tendon. In some embodiments, tendon whichmay be treated by compositions and methods of the present invention isnot selected from the group consisting of supraspinatus tendon,infraspinatus tendon, subscapularis tendon, teres minor tendon (rotatorcuff complex), flexor tendon, rectus femoris tendon, tibialis posteriortendon, and quadriceps femoris tendon.

In some embodiments, the ligament which may be treated by compositionsand methods of the present invention is selected from the groupconsisting of anterior cruciate ligament, lateral collateral ligament,posterior cruciate ligament, medial collateral ligament, cranialcruciate ligament, caudal cruciate ligament, cricothyroid ligament,periodontal ligament, suspensory ligament of the lens, anteriorsacroiliac ligament, posterior sacroiliac ligament, sacrotuberousligament, sacrospinous ligament, inferior pubic ligament, superior pubicligament, suspensory ligament, palmar radiocarpal ligament, dorsalradiocarpal ligament, ulnar collateral ligament, and radial collateralligament.

In some embodiments, bones which may be treated by compositions andmethods of the present invention include, but are not limited to, forexample, tibia, femur, and humerus.

In some embodiments, the tendon or ligament injury not involving a boneis a tendon or ligament rupture, severance, tearing, delamination,strain, or deformation.

In some embodiments, the attachment of the tendon to the bone is forrotator cuff injury treatment. In some embodiments, the attachment ofthe tendon to the bone or ligament to the bone is for anterior cruciateligament reconstruction.

In some embodiments, provided is a method for treatment of a tendon or aligament injury in an individual comprising administering to saidindividual an effective amount of a composition comprising abiocompatible matrix and platelet-derived growth factor (PDGF), whereinthe PDGF is at a concentration in the range of about 0.1 mg/ml to about1.0 mg/ml; and wherein the biocompatible matrix forms a porous structurecomprising pores with a pore area size ranging from about 4500 μm² toabout 20000 μm² and a pore perimeter size ranging from about 200 μm toabout 500 μm.

In some embodiments, provided is a method for treatment of a tendon or aligament injury in an individual comprising administering to an affectedsite of the injury of said individual an effective amount of acomposition comprising a biocompatible matrix and platelet-derivedgrowth factor (PDGF), wherein the PDGF is at a concentration in therange of about 0.1 mg/ml to about 1.0 mg/ml; and wherein the compositionis flowable.

In some embodiments, provided is a method for attaching a tendon into abone or a ligament into a bone in an individual comprising administeringto said individual an effective amount of a composition comprising abiocompatible matrix and platelet-derived growth factor (PDGF) at aninterface between the tendon and the bone or the ligament and the bone,wherein the biocompatible matrix forms a porous structure comprisingpores with a porosity greater than about 85%.

In some embodiments, provided is a method for attaching a tendon into abone in an individual for anterior cruciate ligament reconstructioncomprising administering to said individual an effective amount of acomposition comprising a biocompatible matrix and platelet-derivedgrowth factor (PDGF) at an interface between the tendon and the bone,wherein the biocompatible matrix forms a porous structure comprisingpores with a porosity greater than about 85%.

In some embodiments of the present invention, the method may beperformed using arthroscopic techniques, endoscopic techniques,laparoscopic techniques, or any other suitable minimally-invasivetechniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the cumulative rhPDGF-BB released from different collagenmatrices over 24 hours.

FIG. 2 depicts the percentage of rhPDGF-BB released from differentcollagen matrices over 24 hours.

FIG. 3 depicts the biopotency of rhPDGF-BB after release from thedifferent collagen matrices.

FIG. 4 depicts an exemplary suture-stabilized transected Achillestendon.

FIG. 5 depicts an exemplary administration of a composition to astabilized injured Achilles tendon in accordance with an embodiment ofthe present invention.

FIGS. 6A-6B show the cyclic preconditioning results (conditioningelongation and peak-to-peak elongation) of the Achilles Tendon RepairTreatment A) including two hematoma affected specimens and B) excludingtwo hematoma affected specimens.

FIG. 7A-7B shows the ramp to failure testing results (ultimate force atconstruct failure and global construct stiffness) of the Achilles TendonRepair Treatment.

FIG. 8 shows the repair tissue stiffness results of the Achilles TendonRepair Treatment including or excluding the two hematoma affectedspecimens (left panel and right panel, respectively).

FIGS. 9A-9D depict histological sections of implanted Collagen A (panela), B (panel b), C (panel c), and D (panel d) at 3 weekspost-implantation. Key: B=bone; C=collagen; arrow indicates undegradedcollagen. Scale bar=200 m

FIG. 10 depicts detachment of the long digital extensor tendon, wrappingof the long digital extensor tendon with a collagen sponge, threadingthrough the tibial bone tunnel, and attachment to the medial cortex.Picture is taken from Rodeo S. A. et al., Am. J. Sports Med. 27:476-488(1999).

FIGS. 11A-11C depict side view of the surgical procedure including theformation of a tunnel through the tibial metaphysis. Picture is takenfrom Rodeo S. A. et al., J. Bone Joint Surg. Am., 75(12): 1795-1803(1993).

FIG. 12 depicts orientation of histological specimens. Each block willbe divided equally longitudinally through the bone tunnel and used tocut cross sections and full length, longitudinal sections.

FIG. 13 depicts microtomography (micro CT) of a specimen. Picture istaken from Rodeo S. A. et al., J. Bone Joint Surg. Am., 75(12):1795-1803 (1993).

FIG. 14 depicts a Bench Top Arthroscopic model.

FIG. 15 shows SEM images of ovine tenocytes seeded BIOBLANKET™ matrices5% (panel a), 6% (panel b), 7% (panel c), and COLLATAPE® (panel d) withaddition of rhPDGF-BB in the culture medium at final concentration of 30ng/ml.

FIG. 16 shows the Dynamic Preconditioning Results of the Rotator CuffInjury Treatment. FIG. 16A: The 0.15 mg/ml rhPDGF-BB and 0.30 mg/mlrhPDGF-BB groups underwent significantly greater conditioning elongationthan the Suture Only and Suture+Collagen Matrix groups. FIG. 16B: Therewere no significant differences in peak-to-peak elongation between anygroups.

FIG. 17 shows the Ramp to Failure Results of the Rotator Cuff InjuryTreatment. FIG. 17A: Repair augmentation with 0.15 mg/ml and 0.30 mg/mlrhPDGF-BB resulted in a 63.7% and 63.3% increase in load to failurerelative to the Suture Only group, respectively. FIG. 17B: lowerrhPDGF-BB doses of 0.15 mg/ml and 0.30 mg/ml outperformed the higher 1.0mg/ml PDGF dose, manifested as a 120% and 119.3% increase, respectively,in load at failure.

FIG. 18 is a graphical representation of histopathological scoresgrouped by treatment of 1) Suture+Collagen Matrix+0.15 mg/ml rhPDGF-BBor 2) Suture+Collagen Matrix+0.3 mg/ml rhPDGF-BB. Means are shown anderror bars represent standard deviations.

FIG. 19 is a graphical representation of histopathological scoresgrouped by treatment of 1) Suture only; 2) Suture+Collagen Matrixacetate buffer or 3) Suture+Collagen Matrix+1.0 mg/ml rhPDGF-BB. Meansare shown and error bars represent standard deviations.

FIG. 20 shows region of interdigitation of tendon collagen (box) withbone at the fibrocartilage interface (arrows). A: suture+collagenmatrix, 20×. B: suture+collagen matrix+0.3 mg/ml (or 150 μg) rhPDGF-BB,20×.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the observation thatbiocompatible matrices comprising platelet-derived growth factor (PDGF)may be used in treatment of tendon or ligament injuries not involvingbone and for tendon-bone and ligament-bone tissue repair.

The present invention will use, unless otherwise indicated, conventionaltechniques of molecular biology (including recombinant techniques), cellbiology, biochemistry, nucleic acid chemistry, and immunology, which arewell known to those skilled in the art. The present invention will alsouse, unless otherwise indicated, conventional techniques and apparatusof surgery and other medical methods, which are well known to thoseskilled in the art. Unless defined otherwise, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

As used herein, unless otherwise specified, the term “treatment” or“treating” refers to administrating to an individual an effective amountof a composition comprising a biocompatible matrix and platelet-derivedgrowth factor which alleviates, slows progression of, speeds healing of,improves the healing response of, or repairs a pathology for which theindividual is being treated, and/or which results in one or moredesirable clinical or therapeutic effects which include, but are notlimited to, alleviation of pain associated with an injured tendon orligament, increase in range of motion of the affected joint, andincreased strength and attachment of tendon or ligament to tendon orligament or tendon/ligament to bone at the repair site.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orclinical result. An effective amount can be provided in one or moreadministrations.

As used herein, treatment of “a tendon injury or a ligament injury notinvolving a bone” refers to the treatment of an injured or damagedtendon or ligament that does not involve injured bone or tendon/ligamentdetachment from the bone. Examples of such tendon or ligament injuryinclude, but are not limited to severed tendons/ligaments, rupturedtendons/ligaments, tendons/ligaments exhibiting tearing, delamination,or any other strain or deformation. The individual being treated mayhave injury to a bone or tendon-bone/ligament-bone detachment inaddition to the tendon or ligament injury, however, treatment of “atendon or a ligament injury not involving a bone” refers to the specifictreatment of tendon and/or ligament tissues. It is to be understood thatin some embodiments, an individual may be treated for a tendon injury ora ligament injury not involving a bone as well as an injury involving abone, for example, a tendon-bone or ligament-bone attachment. In someembodiments, only a tendon injury or a ligament injury not involving abone is treated.

An “individual” refers a mammal, including humans, domestic and farmanimals, and zoo, sport, or pet animals, such as chimpanzees and otherapes and monkey species, dogs, horses, rabbits, cattle, pigs, goats,sheep, hamsters, guinea pigs, gerbils, mice, ferrets, rats, cats, andthe like. Preferably, the individual is human. The term does not denotea particular age or gender.

“Bioresorbable” refers to the ability of a biocompatible matrix to beresorbed or remodeled m vivo. The resorption process involvesdegradation and elimination of the original material through the actionof body fluids, enzymes or cells. The resorbed material may be used bythe host in the formation of new tissue, or it may be otherwisere-utilized by the host, or it may be excreted.

Collagen matrices, as referred to herein, include materials in the formof, for example, gels, pastes, particles, powders, sheets, patches,pads, paste, or sponges. Collagen matrices as obtained commercially areusually manufactured from collagen extracts of bovine dermis and/orbovine tendon. In some embodiments, the bovine tendon source is bovinedeep flexor (Achilles) tendon. In some embodiments, the matrices aremade from collagen slurries where the concentration of the collagen inthe slurry is different for each type of matrix. For example, one typeof collagen matrix is made from a slurry with a collagen concentrationof 4.5%, this collagen matrix is referred to herein as “collagen (4.5%)”or “collagen matrix (4.5%)”; a second type of collagen matrix is madefrom a slurry with a collagen concentration of 5%, this collagen matrixis referred to herein as “collagen (5%)” or “collagen matrix (5%)”; athird type of collagen matrix is made from a slurry with a collagenconcentration of 6%, this collagen matrix is referred to herein as“collagen (6%)” or “collagen matrix (6%); and a fourth type of collagenmatrix is made from a slurry with a collagen concentration of 7%, thiscollagen matrix is referred to herein as “collagen (7%)” or “collagenmatrix (7%). For any collagen matrix the percent of collagen used in thestarting slurry does not necessarily reflect the percentage of collagenin the final collagen matrix.

As used herein, the term, “attaching” or “attachment”, as used herein,is meant to include reattaching or reattachment of a tendon to or into abone, or a ligament to or into a bone, and also includes attaching agraft (e.g. a tendon or ligament graft) to or into a bone. For example,using a non-limiting example, when performing anterior cruciate ligamentreconstruction, the injured ligament may be removed from the bone, and agraft, such as a tendon or ligament graft, may be attached to or intothe bone in place of the original ligament.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Compositions and Methods of the Invention

Described herein are compositions and methods for treatment of tendon orligament injuries not involving a bone in an individual. In general, themethods of treatment comprise administering a composition comprising abiocompatible matrix and PDGF to an individual who has a tendon or aligament injury. Specifically, the methods of treatment compriseadministering a composition comprising a collagen matrix and PDGF to thesite of the tendon or the ligament injury.

Described also herein are compositions and methods for attaching orreattaching a tendon or a ligament to/into a bone in an individual. Ingeneral, the methods of attachment comprise administering a compositioncomprising a biocompatible collagen matrix and PDGF at an interfacebetween the tendon/ligament and the bone.

Biocompatible Matrix

A biocompatible matrix, according to some embodiments of the presentinvention, comprises a scaffolding matrix. The scaffolding matrix,according to some embodiments of the present invention, provides aframework or scaffold for new tissue growth to occur, includingtendon/ligament and/or bone tissue.

The biocompatible matrix, in some embodiments, comprises a collagenmatrix. The term “collagen matrix” can refer to, for example, a collagengel, paste, powder, particle, patch, pad, sheet or sponge. In someembodiments, the collagen matrix comprises any type of collagen,including Type I, Type II, and Type III collagens. In some embodiments,the collagen comprises a mixture of collagens, such as a mixture of TypeI and Type II collagen. In other embodiments, the collagen is solubleunder physiological conditions. In some embodiments, the collagen isinsoluble under physiological conditions. In some embodiments, thecollagen comprises soluble and insoluble components. In someembodiments, the collagen matrix comprises a fibrous collagen such assoluble type I bovine collagen. In some embodiments, the collagen matrixcomprises fibrous and acid-soluble collagen derived from bovine dermaltissue. Other types of collagen present in bone or musculoskeletaltissues may be employed. Recombinant, synthetic and naturally occurringforms of collagen may be used in the present invention. Collagen may becross-linked or not cross-linked.

Fibrous collagen suitable for use in the collagen matrix may demonstratesufficient mechanical properties, including wet tensile strength, towithstand suturing and hold a suture without tearing. A fibrous collagenmatrix, for example, can have a wet tear strength ranging from, forexample, about 0.75 pounds to about 5 pounds.

In some embodiments, the biocompatible matrix comprises aglycosaminoglycan (GAG). In some embodiments, the biocompatible matrixcomprises collagen and glycosaminoglycan (GAG), for example cross-linkedcollagen and GAG. In some embodiments, the GAG is chondroitin sulfate.In some embodiments, the GAG is not chondroitin sulfate. Other GAGsinclude, but are not limited to, dermatan sulfate, keratan sulfate,heparin, heparan sulfate, hyaluronan, and combinations thereof. In someembodiments, the weight/weight ratio of collagen to GAG is about 90:10.In some embodiments, the weight/weight ratio of collagen to GAG is about92:8. In some embodiments, the weight/weight ratio of collagen to GAG isabout 95:5. In some embodiments, the biocompatible matrix comprisescross-linked collagen with chondroitin sulfate. In other embodiments,the biocompatible matrix comprises cross-linked collagen with GAG,wherein the GAG is not chondroitin sulfate.

In some embodiments, a collagen matrix is obtained from a commercialsource, including, but not limited to, COLLATAPE® (Integra LifeSciencesCorporation, Plainsboro, N.J.); INTEGRA Flowable Wound Matrix (IntegraLifeSciences Corporation, Plainsboro, N.J.); Cellerate RX, HyCureHydrolyzed Collagen and Hydrolyzed Collagen/Ag Wound Gel (Hymed GroupCorporation, Bethlehem, Pa.); and BIOBLANKET™ and P 1076 flowablecollagen (Kensey Nash Corporation, Exton, Pa.). In some embodiments, thecollagen matrix is not BIOBLANKET™. In some embodiments, the collagenmatrix is not COLLATAPE®.

In some embodiments, the biocompatible matrix of the present inventionis flowable. Flowable biocompatible matrix, in some embodiments, can beapplied to the desired site through a syringe and needle or cannula. Insome embodiments, the flowable biocompatible matrix can be applied tothe desired site percutaneously. In other embodiments, flowablebiocompatible matrix can be applied to a surgically exposed site. Insome embodiments, flowable biocompatible matrix can be applied to atendon or ligament graft for insertion into a bone tunnel. In someembodiments, such as in a kit, the biocompatible matrix is provided as adehydrated powder or particles, which can be made flowable uponpreparation for administration. For example, a dehydrated form of thebiocompatible matrix may be prepared for administration by addition toor mixing with a suitable amount of a hydrating buffer. The hydratingbuffer may include a suitable amount of PDGF to be administered as partof the biocompatible matrix. The suitable amount of buffer to be addedis determined by, for example, the desired concentration of PDGF, thedesired concentration of collagen, the desired concentration of GAG, thedesired flowability characteristic, or any suitable combination thereof.

“Flowable” refers to a physical characteristic of a substance in whichthe substance flows upon application of a force required to administersuch substance through a cannula or like passage, yet the substance willremain substantially immobile after administration to a site in theindividual to be treated, thereby providing continued treatment to thesite. An exemplary device for administering a flowable substance is asyringe, in which the plunger can provide the required force that urgesthe flowable substance to be administered. A suitable exemplary devicemay further comprise a needle or other suitable cannula that allow moreprecise delivery and application of the flowable substance. The borediameter, the composition, the conformation, the length, and any othersuitable characteristic of the device for administration may be selectedand used. A person skilled in the art understands that the selection ofparticular parameters of a suitable delivery device are based on theflowability characteristics of the composition. For example, relativelyless flowable composition may be more suited for delivery by a devicehaving a relatively wider cannular bore diameter and/or shorter cannula.In some embodiments, the flowable composition is delivered through a 21G, 25 G, or 27 G needle. In some embodiments, delivery by syringe and/orneedle allows delivery of the flowable composition via percutaneousinjection. In some embodiments, the flowable composition can bedelivered by a non-cannular device, such as a scoop, spatula, brush, orother like device, which allows the composition to be delivered to thedesired location.

In some embodiments, the biocompatible matrix does not oxidize PDGF.

Biocompatible matrix, according to some embodiments, can be provided ina shape suitable for implantation (e.g., a sphere, a cylinder, or ablock). In other embodiments, biocompatible matrix is moldable orextrudable. In such embodiments, the biocompatible matrix can be in theform of a paste or putty. In some embodiments, the biocompatible matrixcan be provided in a predetermined shape including a block, sphere, orcylinder, or any desired shape, for example a shape defined by a mold ora site of application. Moldable biocompatible matrix can facilitateefficient placement of compositions of the present invention in andaround tendons, ligaments, and/or bone, including sites of tendon orligament attachment into or to bone, such as insertion of a tendon orligament graft into a bone tunnel. In some embodiments, moldablebiocompatible matrix can be applied to bone and/or tendons and/orligaments with a spatula or equivalent device. In some embodiments,biocompatible matrix can be applied to tendons or ligaments by wrappingaround the tendons or ligaments, such as tendon or ligament grafts.

Collagen matrices of the present invention can be made from purifiedcollagen extracts from bovine dermis, bovine tendon (e.g., deep flexor(Achilles) tendon), or other suitable collagen sources. In someembodiments, the collagen matrix is primarily Type I collagen. In someembodiments the collagen matrix is made from a collagen slurry with anyone of the following concentrations of collagen (w/v): about 4.5%, about5%, about 6% or about 7%. In some embodiments, the collagen is soluble.A soluble collagen can dissolve shortly after its implantation, therebyintroducing macroporosity into the biocompatible matrix. Macroporositycan increase the conductivity of cells (e.g. osteoblast, tenocyte) intothe implant material by enhancing the access and, consequently, theremodeling activity of the cells at the implant site.

In some embodiments, the collagen comprises a soluble collagen monomercomponent and an insoluble collagen polymer component. In some embodies,the ratio of soluble collagen monomers to insoluble collagen polymers isabout 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about1:4, about 1:3, about 1:2 or about 1:1.

In some preferred embodiments, the biocompatible matrix forms a porousstructure comprising pores. In some embodiments, the pores have anaverage area ranging from about 800 um² to about 3,000 um², about 13,000um² to about 50,000 um², about 2500 um² to about 20,000 um², about 3500um² to about 20,000 um², 4500 um² to about 20,000 um², about 5000 um² toabout 19,000 um², about 6000 um² to about 18,000 um², about 6000 um² toabout 15,000 um², or about 5000 um² to about 16000 um². In someembodiments, the pores have an average perimeter ranging from about 100μm to about 200 μm, about 400 μm to about 800 μm, about 200 μm to about500 μm, about 200 jam to about 600 μm, about 300 μm to about 600 μm, orabout 300 μm to about 500 μm. In some embodiments, the biocompatiblematrix has an average pore area size ranging from about 4500 μm² toabout 20000 μm² and an average pore perimeter size ranging from about200 μm to about 600 μm. In some embodiments, the collagen matrixcomprises pores with an average pore area size ranging from about 4500μm² to about 20000 μm² and an average pore perimeter size ranging fromabout 200 μm to about 500 μm.

In some embodiments, the biocompatible matrix comprises a porousstructure having multidirectional and/or interconnected pores. Porousstructure, according to some embodiments, can comprise pores havingdiameters ranging from about 1 μm to about 1 mm, for example, at leastabout 5 μm, at least about 10 μm, at least about 20 μm, at least about30 um, at least about 40 μm, or at least about 50 μm.

In some embodiments, the biocompatible matrix comprises macroporeshaving diameters ranging from about 100 μm to about 1 mm. In anotherembodiment, the biocompatible matrix comprises mesopores havingdiameters ranging from about 10 μm to about 100 μm. In a furtherembodiment, the biocompatible matrix comprises micropores havingdiameters less than about 10 μm. Embodiments of the present inventioncontemplate biocompatible matrix comprising macropores, mesopores, andmicropores or any combination thereof.

In other embodiments, the biocompatible matrix comprises a porousstructure having pores that are not interconnected. In some embodiments,the biocompatible matrix comprises a porous structure having a mixtureof interconnected pores and pores that are not interconnected.

In some embodiments, the biocompatible matrix is porous and able toabsorb water in an amount ranging from about 1× to about 15× the mass ofthe biocompatible matrix. In some embodiments, the collagen matrix isporous and able to absorb water in an amount ranging from about 1× toabout 15× the mass of the collagen matrix.

In some embodiments, the porous structure of the biocompatible matrixallows for PDGF to be released from the matrix in increased amounts. Insome embodiments, the porous structure of collagen matrix (5%) releasesa higher percentage of PDGF than collagen matrix (6%) or collagen matrix(7%) within a particular time, as measured in vivo or in vitro. In someembodiments, the porous structure of the biocompatible matrix allows forPDGF to be released from the matrix within about 24 hours of in vivo orin vitro administration. In some embodiments, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, about 50% to about 95%, about 60%to about 95%, about 70% to about 95%, about 80% to about 95%, about 90%to about 95%, about 50% to about 85%, about 60% to about 85%, about 70%to about 85%, or about 50% to about 80% of the PDGF is released from thematrix within about 24 hours. In some embodiments, the porous structureof the biocompatible matrix allows for PDGF to be released from thematrix within about 1 hour, about 6 hours, about 8 hours, about 12hours, or about 48 hours of in vivo or in vitro administration. In someembodiments, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,about 50% to about 95%, about 60% to about 95%, about 70% to about 95%,about 80% to about 95%, about 90% to about 95%, about 50% to about 85%,about 60% to about 85%, about 70% to about 85%, or about 50% to about80% of the PDGF is released within about 1 hour, about 6 hours, about 8hours, about 12 hours, or about 48 hours. In some embodiments, thecollagen matrix is COLLATAPE®. In some embodiments, the collagen matrixis a porous bovine collagen sponge similar to COLLATAPE®. In someembodiments, the biocompatible matrix comprises COLLATAPE® which allowsfor a higher percentage of PDGF to be released in comparison to collagenmatrix (5%), collagen matrix (6%), or collagen matrix (7%).

In some embodiments, the PDGF is released into the surrounding region.In some embodiments, the PDGF is released onto an injured tendon orligament. In some embodiments, the PDGF is released onto the surface ofthe bone near the point of bone-tendon or bone-ligament attachment. Insome embodiments, the PDGF is released into the surrounding media.

In some embodiments, the porous structure of the biocompatible matrixallows for infiltration of cells into pores of the matrix. In someembodiments, the cells infiltrate the composition within about 3 weeks,about 2 weeks, about 1 week, about 7 days, about 6 days, about 5 days,about 4, about 3, about 2, or about 1 day(s) after exposure to thecomposition. In some embodiments, the cells are tenocytes. In someembodiments the cells are osteoblasts. In some embodiments, the cellsare ligament cells. In preferred embodiments, the biocompatible matrixcomprises COLLATAPE® with a porous structure that allows forinfiltration of cells into the pores of the matrix. In some embodiments,the biocompatible matrix comprises COLLATAPE® with a porous structurethat allows for a larger number of cells to infiltrate into the pores ofthe matrix as compared to collagen matrix (5%), collagen matrix (6%), orcollagen matrix (7%). In some embodiments, the biocompatible matrixcomprises collagen matrix (5%) with a porous structure that allows for alarger number of cells to infiltrate into the pores of the matrix ascompared to collagen matrix (6%), or collagen matrix (7%).

In some embodiments, the biocompatible matrix has a porosity of at leastabout 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about98%, or about 99%.

In some embodiments, a biocompatible matrix is bioresorbable. Abiocompatible matrix, in some embodiments, can be resorbed within oneyear of in vivo administration. In other embodiments, a biocompatiblematrix can be resorbed within 1, 3, 6, or 9 months of in vivoadministration. In some embodiments, the biocompatible matrix isresorbed within about 30 days, about 25 days, about 21 days, about 18days, about 15 days, about 10-14 days, or about 10 days of in vivoadministration. Bioresorbability is dependent on: (1) the nature of thematrix material (i.e., its chemical make up, physical structure andsize); (2) the location within the body in which the matrix is placed;(3) the amount of matrix material that is used; (4) the metabolic stateof the patient (diabetic/non-diabetic, osteoporotic, smoker, old age,steroid use, etc.); (5) the extent and/or type of injury treated; and(6) the use of other materials in addition to the matrix such as otherbone anabolic, catabolic and anti-catabolic factors.

In some embodiments, the biocompatible matrix comprises at least onecalcium phosphate. In other embodiments, the biocompatible matrixcomprises a plurality of calcium phosphates. In some embodiments,calcium phosphates suitable for use as a biocompatible matrix material,have a calcium to phosphorus atomic ratio ranging from 0.5 to 2.0. Insome embodiments, the biocompatible matrix comprises an allograft. Insome embodiments, the biocompatible matrix is for use in treating atendon or ligament injury not involving a bone, and the matrix does notcomprise a calcium phosphate or an allograft.

Calcium phosphates suitable for use as bone scaffolding materialsinclude, but are not limited to amorphous calcium phosphate, monocalciumphosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA),dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous(DCPA), octacalcium phosphate (OCP), α-tricalcium phosphate, β-TCP,hydroxyapatite (OHAp), poorly crystalline hydroxyapatite, tetracalciumphosphate (TTCP), heptacalcium decaphosphate, calcium metaphosphate,calcium pyrophosphate dehydrate, carbonated calcium phosphate, andcalcium pyrophosphate.

Scaffolding Material and Biocompatible Binder

In some embodiments, the biocompatible matrix comprises a scaffoldingmatrix and a biocompatible binder. Biocompatible binders can comprisematerials operable to promote cohesion between combined substances. Abiocompatible binder, for example, can promote adhesion betweenparticles of a bone scaffolding material in the formation of abiocompatible matrix. In certain embodiments, the same material mayserve as both a scaffolding material and a binder if such material actsto promote cohesion between the combined substances and provides aframework for new tissue growth to occur, including tendon, ligament,and bone growth. See WO2008/005427 and U.S. Ser. No. 11/772,646 (U.S.Publication 2008/00274470).

Biocompatible binders, in some embodiments, can comprise, for example,collagen, elastin, polysaccharides, nucleic acids, carbohydrates,proteins, polypeptides, poly(α-hydroxy acids), poly(lactones),poly(amino acids), poly(anhydrides), polyurethanes, poly(orthoesters),poly(anhydride-co-imides), poly(orthocarbonates), poly(α-hydroxyalkanoates), poly(dioxanones), poly(phosphoesters), polylactic acid,poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D,L-lactide),poly(D,L-lactide-co-trimethylene carbonate), polyglycolic acid,polyhydroxybutyrate (PHB), poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(caprolactone), polyacrylic acid,polycarboxylic acid, poly(allylamine hydrochloride),poly(diallyldimethylammonium chloride), poly(ethyleneimine),polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,polyethylene, polymethylmethacrylate, carbon fibers, poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and/or copolymers and/or mixtures thereof.

Biocompatible binders, in other embodiments, can comprise alginic acid,arabic gum, guar gum, xantham gum, gelatin, chitin, chitosan, chitosanacetate, chitosan lactate, chondroitin sulfate, N,O-carboxymethylchitosan, a dextran (e.g., α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or sodium dextran sulfate), fibrin glue, lecithin,phosphatidylcholine derivatives, glycerol, hyaluronic acid, sodiumhyaluronate, a cellulose (e.g., methylcellulose, carboxymethylcellulose,hydroxypropyl methylcellulose, or hydroxyethyl cellulose), aglucosamine, a proteoglycan, a starch (e.g., hydroxyethyl starch orstarch soluble), lactic acid, a pluronic acids, sodium glycerophosphate,glycogen, a keratin, silk, and/or derivatives and/or mixtures thereof.

In some embodiments, a biocompatible binder is water-soluble. Awater-soluble binder can dissolve from the biocompatible matrix shortlyafter its implantation, thereby introducing macroporosity into thebiocompatible matrix. Macroporosity can increase the osteoconductivityof the implant material by enhancing the access and, consequently, theremodeling activity of the cells (e.g. osteoclasts and osteoblasts,tenocytes) at the implant site.

In some embodiments, a biocompatible binder can be present in abiocompatible matrix in an amount ranging from about 5 weight percent toabout 50 weight percent of the matrix. In other embodiments, abiocompatible binder can be present in an amount ranging from about 10weight percent to about 40 weight percent of the biocompatible matrix.In another embodiment, a biocompatible binder can be present in anamount ranging from about 15 weight percent to about 35 weight percentof the biocompatible matrix. In a further embodiment, a biocompatiblebinder can be present in an amount of about 20 weight percent of thebiocompatible matrix.

Platelet-Derived Growth Factor

The invention provides for compositions and methods for the treatment oftendon or ligament injuries in an individual. In general, the methods oftreatment comprise administering a composition comprising abiocompatible matrix and PDGF to an individual who has a tendon orligament injury. Specifically, the methods of treatment compriseadministering a composition comprising a collagen matrix and PDGF to thesite of the tendon or ligament injury.

A biocompatible matrix, according to embodiments of the presentinvention, comprises a scaffolding matrix and PDGF. PDGF is a growthfactor released from platelets at sites of injury. PDGF synergizes withVEGF to promote neovascularization, and stimulates chemotaxis andproliferation of mesenchymally-derived cells including tenocytes,osteoblasts, chondrocytes and vascular smooth muscle cells.

Compositions and methods provided by the present invention comprise abiocompatible matrix and a solution of PDGF wherein the solution isdispersed in the biocompatible matrix. In some embodiments, PDGF ispresent in the solution in a concentration ranging from about 0.01 mg/mlto about 10.0 mg/ml, from about 0.05 mg/ml to about 5.0 mg/ml, fromabout 0.1 mg/ml to about 1.0 mg/ml, or from about 0.1 mg/ml to about 2.0mg/ml, about 0.1 mg/ml to about 0.4 mg/ml, about 0.9 mg/ml to about 1.5mg/ml. In some embodiments, the PDGF is present in the solution at aconcentration of 0.15 mg/ml. In some embodiments, the PDGF is present inthe solution at a concentration of 0.3 mg/ml. In some embodiments, thePDGF is present in the solution at a concentration of 1.0 mg/ml. Inother embodiments, PDGF is present in the solution at any one of thefollowing concentrations: about 0.05 mg/ml; about 0.1 mg/ml; about 0.15mg/ml; about 0.2 mg/ml; about 0.25 mg/ml; about 0.3 mg/ml; about 0.35mg/ml; about 0.4 mg/ml; about 0.45 mg/ml; about 0.5 mg/ml, about 0.55mg/ml, about 0.6 mg/ml, about 0.65 mg/ml, about 0.7 mg/ml; about 0.75mg/ml; about 0.8 mg/ml; about 0.85 mg/ml; about 0.9 mg/ml; about 0.95mg/ml; about 1.0 mg/ml; about 1.5 mg/ml; or about 2.0 mg/ml. It is to beunderstood that these concentrations are simply examples of particularembodiments, and that the concentration of PDGF may be within any of theconcentration ranges stated above.

Various amounts of PDGF may be used in the compositions of the presentinvention. Amounts of PDGF that can be used include, but are not limitedto, amounts in the following ranges: about 1 μg to about 50 mg, about 10μg to about 25 mg, about 100 μg to about 10 mg, and about 250 μg toabout 5 mg.

The concentration of PDGF (or other growth factors) in embodiments ofthe present invention can be determined by using an enzyme-linkedimmunoassay as described in U.S. Pat. Nos. 6,221,625; 5,747,273; and5,290,708, or any other assay known in the art for determining PDGFconcentration. When provided herein, the molar concentration of PDGF isdetermined based on the molecular weight of PDGF dimer (e.g., PDGF-BB,MW about 25 kDa).

In some embodiments of the present invention, PDGF comprises PDGFhomodimers and heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB,PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In someembodiments, PDGF comprises PDGF-BB. In other embodiments, PDGFcomprises a recombinant human PDGF, such as rhPDGF-BB.

In some embodiments, PDGF can be obtained from natural sources. In otherembodiments, PDGF can be produced by recombinant DNA techniques. In someembodiments, PDGF or fragments thereof may be produced using peptidesynthesis techniques known to one of skill in the art, such as solidphase peptide synthesis.

When obtained from natural sources, PDGF can be derived from biologicalfluids. Biological fluids, according to some embodiments, can compriseany treated or untreated fluid associated with living organismsincluding blood. Biological fluids can also comprise blood componentsincluding platelet concentrate, apheresed platelets, platelet-richplasma, plasma, serum, fresh frozen plasma, and buffy coat. Biologicalfluids can comprise platelets separated from plasma and resuspended in aphysiological fluid.

When produced by recombinant DNA techniques, a DNA sequence encoding asingle monomer (e.g., PDGF B-chain or A-chain) can be inserted intocultured prokaryotic or eukaryotic cells for expression to subsequentlyproduce the homodimer (e.g., PDGF-BB or PDGF-AA). The homodimer PDGFproduced by recombinant techniques may be used in some embodiments. Inother embodiments, a PDGF heterodimer can be generated by inserting DNAsequences encoding for both monomeric units of the heterodimer intocultured prokaryotic or eukaryotic cells and allowing the translatedmonomeric units to be processed by the cells to produce the heterodimer(e.g., PDGF-AB). Commercially available recombinant human PDGF-BB may beobtained from a variety of sources.

In some embodiments of the present invention, PDGF comprises PDGFfragments. In one embodiment, rhPDGF-B comprises the followingfragments: amino acid sequences 1-31, 1-32, 33-108, 33-109, and/or 1-108of the entire B chain. The complete amino acid sequence (aa 1-109) ofthe B chain of PDGF is provided in FIG. 15 of U.S. Pat. No. 5,516,896.It is to be understood that the rhPDGF compositions of the presentinvention may comprise a combination of intact rhPDGF-B (aa 1-109) andfragments thereof. Other fragments of PDGF may be employed such as thosedisclosed in U.S. Pat. No. 5,516,896. In accordance with someembodiments, the rhPDGF-BB comprises at least 65% of intact rhPDGF-B (aa1-109). In accordance with other embodiments, the rhPDGF-BB comprises atleast 75%, 80%, 85%, 90%, 95%, or 99% of intact rhPDGF-B (aa 1-109).

In some embodiments of the present invention, PDGF can be in a highlypurified form. Purified PDGF, as used herein, comprises compositionshaving greater than about 95% by weight PDGF prior to incorporation insolutions of the present invention. The solution may be prepared usingany pharmaceutically acceptable buffer or diluent. In other embodiments,the PDGF can be substantially purified. Substantially purified PDGF, asused herein, comprises compositions having about 5% to about 95% byweight PDGF prior to incorporation into solutions of the presentinvention. In one embodiment, substantially purified PDGF comprisescompositions having about 65% to about 95% by weight PDGF prior toincorporation into solutions of the present invention. In otherembodiments, substantially purified PDGF comprises compositions havingabout 70% to about 95%, about 75% to about 95%, about 80% to about 95%,about 85% to about 95%, or about 90% to about 95%, by weight PDGF, priorto incorporation into solutions of the present invention. Purified PDGFand substantially purified PDGF may be incorporated into the scaffoldingmatrix.

In a further embodiment, PDGF can be partially purified. Partiallypurified PDGF, as used herein, comprises compositions having PDGF in thecontext of platelet-rich plasma, fresh frozen plasma, or any other bloodproduct that requires collection and separation to produce PDGF.Embodiments of the present invention contemplate that any of the PDGFisoforms provided herein, including homodimers and heterodimers, can bepurified or partially purified. Compositions of the present inventioncomprising PDGF mixtures may comprise PDGF isoforms or PDGF fragments inpartially purified proportions. Partially purified and purified PDGF, insome embodiments, can be prepared as described in U.S. Ser. No.11/159,533 (U.S. Publication 20060084602).

In some embodiments, solutions comprising PDGF are formed bysolubilizing PDGF in one or more buffers. Buffers suitable for use inPDGF solutions of the present invention can comprise, but are notlimited to, carbonates, phosphates (e.g. phosphate-buffered saline),histidine, acetates (e.g. sodium acetate), acidic buffers such as aceticacid and HCl, and organic buffers such as lysine, Tris buffers (e.g.tris(hydroxymethyl)aminoethane),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be selectedbased on biocompatibility with PDGF and the buffer's ability to impedeundesirable protein modification. Buffers can additionally be selectedbased on compatibility with host tissues. In one embodiment, sodiumacetate buffer is used. The buffers may be employed at differentmolarities, for example about 0.1 mM to about 100 mM, about 1 mM toabout 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, orabout 15 mM to about 25 mM, or any molarity within these ranges. In someembodiments, an acetate buffer is employed at a molarity of about 20 mM.

In another embodiment, solutions comprising PDGF may be formed bysolubilizing lyophilized PDGF in water, wherein prior to solubilizationthe PDGF is lyophilized from an appropriate buffer.

Solutions comprising PDGF, according to embodiments of the presentinvention, can have a pH ranging from about 3.0 to about 8.0. In oneembodiment, a solution comprising PDGF has a pH ranging from about 5.0to about 8.0, more preferably about 5.5 to about 7.0, most preferablyabout 5.5 to about 6.5, or any value within these ranges. The pH ofsolutions comprising PDGF, in some embodiments, can be compatible withthe prolonged stability and efficacy of PDGF or any other desiredbiologically active agent. PDGF is generally more stable in an acidicenvironment. Therefore, in accordance with some embodiments, the presentinvention comprises an acidic storage formulation of a PDGF solution. Inaccordance with some embodiments, the PDGF solution preferably has a pHfrom about 3.0 to about 7.0, and more preferably from about 4.0 to about6.5. The biological activity of PDGF, however, can be optimized in asolution having a neutral pH range. Therefore, in other embodiments, thepresent invention comprises a neutral pH formulation of a PDGF solution.In accordance with this embodiment, the PDGF solution preferably has apH from about 5.0 to about 8.0, more preferably about 5.5 to about 7.0,most preferably about 5.5 to about 6.5.

In some embodiments, the pH of the PDGF-containing solution may bealtered to optimize the binding kinetics of PDGF to a matrix substrate.If desired, as the pH of the material equilibrates to adjacent material,the bound PDGF may become labile. The pH of solutions comprising PDGF,in some embodiments, can be controlled by the buffers recited herein.Various proteins demonstrate different pH ranges in which they arestable. Protein stabilities are primarily reflected by isoelectricpoints and charges on the proteins. The pH range can affect theconformational structure of a protein and the susceptibility of aprotein to proteolytic degradation, hydrolysis, oxidation, and otherprocesses that can result in modification to the structure and/orbiological activity of theprotein.

In some embodiments, solutions comprising PDGF can further compriseadditional components, such as other biologically active agents. Inother embodiments, solutions comprising PDGF can further comprise cellculture media, other stabilizing proteins such as albumin, antibacterialagents, protease inhibitors (e.g., ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(beta-aminoethylether)-N,N,N′,N′-tetraaceticacid (EGTA), aprotinin, E-aminocaproic acid (EACA), etc.) and/or othergrowth factors such as fibroblast growth factors (FGFs), epidermalgrowth factors (EGFs), transforming growth factors (TGFs), keratinocytegrowth factors (KGFs), insulin-like growth factors (IGEs), bonemorphogenetic proteins (BMPs), or other PDGFs including compositions ofPDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or PDGF-DD.

Compositions Further Comprising Biologically Active Agents

Compositions and methods of the present invention, according to someembodiments, can further comprise one or more biologically active agentsin addition to PDGF. Biologically active agents that can be incorporatedinto compositions of the present invention, in addition to PDGF, cancomprise organic molecules, inorganic materials, proteins, peptides,nucleic acids (e.g., genes, gene fragments, small-interferingribonucleic acids (siRNAs), gene regulatory sequences, nucleartranscriptional factors and antisense molecules), nucleoproteins,polysaccharides (e.g., heparin), glycoproteins, and lipoproteins.Non-limiting examples of biologically active compounds that can beincorporated into compositions of the present invention, including,e.g., anti-cancer agents, antibiotics, analgesics, anti-inflammatoryagents, immunosuppressants, enzyme inhibitors, antihistamines, hormones,muscle relaxants, prostaglandins, trophic factors, osteoinductiveproteins, growth factors, and vaccines, are disclosed in U.S. Ser. No.11/159,533 (U.S. Publication 20060084602). Biologically active compoundsthat can be incorporated into compositions of the present invention, insome embodiments, include osteoinductive factors such as insulin-likegrowth factors, fibroblast growth factors, or other PDGFs. In accordancewith other embodiments, biologically active compounds that can beincorporated into compositions of the present invention preferablyinclude osteoinductive and osteostimulatory factors such as bonemorphogenetic proteins (BMPs), BMP mimetics, calcitonin, calcitoninmimetics, statins, statin derivatives, fibroblast growth factors,insulin-like growth factors, growth differentiating factors, and/orparathyroid hormone. Additional factors for incorporation intocompositions of the present invention, in some embodiments, includeprotease inhibitors, as well as osteoporotic treatments that decreasebone resorption including bisphosphonates, and antibodies to the NF-kB(RANK) ligand.

Standard protocols and regimens for delivery of additional biologicallyactive agents are known in the art. Additional biologically activeagents can be introduced into compositions of the present invention inamounts that allow delivery of an appropriate dosage of the agent to thedamaged tendon and/or the site of tendon attachment. In most cases,dosages are determined using guidelines known to practitioners andapplicable to the particular agent in question. The amount of anadditional biologically active agent to be included in a composition ofthe present invention can depend on such variables as the type andextent of the condition, the overall health status of the particularpatient, the formulation of the biologically active agent, releasekinetics, and the bioresorbability of the biocompatible matrix. Standardclinical trials may be used to optimize the dose and dosing frequencyfor any particular additional biologically active agent.

A composition for attaching tendon or ligament to or into bone accordingto some embodiments, further comprises one or more bone graftingmaterials including, for example, autologous bone marrow, autologousplatelet extracts, allografts, synthetic bone matrix materials,xenografts, and derivatives thereof.

Tendons, Ligaments, and Bones Treated

Tendons which may be treated by the methods of the invention include anytendon. Non-limiting examples of tendons include patellar tendon,anterior tibialis tendon, Achilles tendon, Hamstring tendon,semitendinosus tendon, gracilis tendon, abductor tendon, adductortendon, supraspinatus tendon, infraspinatus tendon, subscapularistendon, teres minor tendon (rotator cuff complex), flexor tendon, rectusfemoris tendon, tibialis posterior tendon, and quadriceps femoristendon. In some embodiments, the tendon is selected from the groupconsisting of patellar tendon, anterior tibialis tendon, Achillestendon, Hamstring tendon, semitendinosus tendon, gracilis tendon,abductor tendon, and adductor tendon. In some embodiments, the tendon isselected from the group consisting of supraspinatus tendon,infraspinatus tendon, subscapularis tendon, teres minor tendon (rotatorcuff complex), flexor tendon, rectus femoris tendon, tibialis posteriortendon, and quadriceps femoris tendon. In some embodiments, the tendonis not selected from the group consisting of supraspinatus tendon,infraspinatus tendon, subscapularis tendon, teres minor tendon (rotatorcuff complex), flexor tendon, rectus femoris tendon, tibialis posteriortendon, and quadriceps femoris tendon.

Ligaments which may be treated by the methods of the invention includeany ligaments. Non-limiting examples of ligaments include anteriorcruciate ligament, lateral collateral ligament, posterior cruciateligament, medial collateral ligament, cranial cruciate ligament, caudalcruciate ligament, cricothyroid ligament, periodontal ligament,suspensory ligament of the lens, anterior sacroiliac ligament, posteriorsacroiliac ligament, sacrotuberous ligament, sacrospinous ligament,inferior pubic ligament, superior pubic ligament, suspensory ligament(e.g., penis or breast), palmar radiocarpal ligament, dorsal radiocarpalligament, ulnar collateral ligament, or radial collateral ligament.

Bones which may be treated by compositions and methods of the presentinvention include any bones which are attachment sites for tendons orligaments, and include, but are not limited to, the tibia, femur, andhumerus.

Methods for Treatment of Tendon or Ligament Injuries not Involving aBone

The present invention provides compositions and methods for thetreatment of tendon or ligament injuries. The methods for treatment maycomprise treatment of injured tendons or ligaments not involving a bone,such as severed tendons/ligaments, ruptured tendons/ligaments,tendons/ligaments exhibiting tearing, delamination, or any other strainor deformation.

In some embodiments, a method for treatment of tendon or ligamentinjuries is directed to administering a composition of the presentinvention to an injured tendon or ligament. In some embodiments, theinjured tendon or ligament may be physically stabilized for thetreatment. For example, the injured tendon or ligament may be sutured bya modified Mason Allen suture design, or any other suitable suture. Suchstabilizing methods may be preferred in some embodiments, as the damagedends of the tendon or ligament may not permit direct surgical repair orreconnection of the ends. As a result, the stabilizing suture may bepositioned distal to one or more of the injured ends.

As a result of this stabilizing suture, the injured tendon or ligamentis positioned such that the injured ends of the tendon or ligament aresubstantially re-approximated. In some embodiments, a suitably-sized gapmay remain between the re-approximated ends, thereby allowingintroduction of a flowable composition into the gap volume, therebybridging or filling-in the gap. In some embodiments, the administeredcomposition can be substantially immobile in the gap. In someembodiments, this substantial immobility may be aided or provided by awrap, or other suitable device, to surround or bind the re-approximatedends with the composition, thereby enclosing the injury and thecomposition therein.

The composition of the present invention can be flowable. In thismanner, the flowable composition can be more precisely delivered to theinjured tendon or ligament site. A syringe, or other suitable device,can be used to administer the composition of the present invention. Thesyringe may further include a needle or other suitable cannula thatallows more precise delivery of the composition. The configuration ofthe syringe and/or cannula, such as its bore, size, length, etc. can beconfigured depending on the desired volume of composition to bedelivered, or the flowability characteristics of the composition.Another advantageous characteristic of the composition of the presentinvention is that the composition, once delivered, can remainsubstantially immobile at its delivery site. In some embodiments, theflowable composition is delivered through a 21 G, 25 G, or 27 G needle.In some embodiments, delivery by syringe and/or needle allows deliveryof the flowable composition via percutaneous injection. In someembodiments, the flowable composition can be delivered by a non-cannulardevice, such as a scoop, spatula, brush, or other like device, whichallows the composition to be delivered to the desired location.

Still another advantageous feature of the composition in one embodimentof the present invention is that its flowable characteristics allow thecomposition to access and fill-in regions of the injury that may benormally difficult to access. For example, a ruptured tendon or ligamentmay be significantly frayed at the injured ends, thereby making directsurgical repair difficult. Furthermore, such damaged ends may containmany small, relatively inaccessible regions, such as crevices and otherinterstitial regions. The flowable composition of the present inventioncan be administered so that it will flow into, and thus substantiallyfill-in, such regions, thus allowing and promoting a more effectiverepair and revascularization of the injury.

In some embodiments of the present invention, the method may beperformed using arthroscopic techniques, endoscopic techniques,laparoscopic techniques, or any other suitable minimally-invasivetechniques.

In some embodiments, the method for treatment of a tendon or ligamentinjury not involving a bone in an individual comprises administering toan affected site of the injury of the individual an effective amount ofa composition comprising: a biocompatible matrix and PDGF, wherein thebiocompatible matrix comprises pores, wherein the biocompatible matrixhas a porosity of at least about 80%, and wherein at least about 50% ofthe PDGF is released within about 24 hours. In some embodiments, themethod for treatment of a tendon or ligament injury not involving a bonein an individual comprises administering to the individual an effectiveamount of a composition comprising: a biocompatible matrix and PDGF,wherein the biocompatible matrix comprises pores, wherein thebiocompatible matrix has a porosity of at least about 80%, and whereinat least about 50% of the PDGF is released within about 24 hours,wherein the PDGF is present as a solution comprising PDGF, wherein theconcentration of PDGF in the solution is about 0.1 mg/ml to about 2.0mg/ml. In some embodiments, the concentration of PDGF in the solution isabout 1.0 mg/ml. In some embodiments, the biocompatible matrix comprisesa cross-linked collagen and glycosaminoglycan matrix. In someembodiments, at least about 60% of the PDGF is released within about 24hours. In some embodiments, at least about 70% of the PDGF is releasedwithin about 24 hours. In some embodiments, at least about 80% of thePDGF is released within about 24 hours. In some embodiments, thebiocompatible matrix has a porosity of at least about 85%. In someembodiments, the biocompatible matrix has a porosity of at least about90%. In some embodiments, the biocompatible matrix has a porosity of atleast about 92%. In some embodiments, the composition is flowable. Insome embodiments, the biocompatible matrix is Integra Flowable WoundMatrix. In some embodiment, the treatment is of a tendon. In someembodiments, the treatment is of a ligament.

The present invention also provides methods of treating Achilles tendoninjuries. In one embodiment, a method for treating Achilles tendoninjuries comprises providing a biocompatible matrix and PDGF, whereinthe biocompatible matrix is flowable.

The present invention also provides compositions and methods fortreating any other suitable tendon injury, including, but are notlimited to, tendons of the subscapularis, supraspinatus, infraspinatus,teres minor, rectus femoris, tibialis posterior, quadriceps femoris,biceps brachii, as well as the Achilles Tendon, patellar tendon,abductor and adductor tendons, or other tendons of the hip, the commonextensor tendon, common flexor tendon, flexor digitorum superficialistendons, extensor digitorum and extensor minimi tendons, or othertendons of the arm and hand.

The present invention also provides compositions and methods fortreating any other suitable ligament injury, including, but are notlimited to anterior cruciate ligament, lateral collateral ligament,posterior cruciate ligament, medial collateral ligament, cranialcruciate ligament, caudal cruciate ligament, cricothyroid ligament,periodontal ligament, suspensory ligament of the lens, anteriorsacroiliac ligament, posterior sacroiliac ligament, sacrotuberousligament, sacrospinous ligament, inferior pubic ligament, superior pubicligament, suspensory ligament (e.g., penis or breast), palmarradiocarpal ligament, dorsal radiocarpal ligament, ulnar collateralligament, or radial collateral ligament.

Methods for Tendon to Bone or Ligament to Bone Attachment

The present invention provides methods for attaching or reattaching atendon or a ligament into/to a bone and for strengthening of tendon orligament attachment to bone. The methods for attachment may comprisetendon or ligament integration or reintegration with bone at aninterface between a tendon and a bone and a ligament and a bone. In someembodiments, the methods for attaching a tendon or a ligament to or intoa bone comprises providing a composition comprising a PDGF solutiondisposed in a biocompatible matrix and applying the composition at aninterface between a tendon/ligament and a bone.

In some embodiments, a method for attachment comprises providing acomposition comprising a PDGF solution disposed in a biocompatiblematrix, wrapping around a detached tendon, inserting the composition andthe tendon into a tunnel that is drilled in the bone, and attaching thetendon to the bone with sutures.

In some embodiments, a method for attachment comprises providing acomposition comprising a PDGF solution disposed in a biocompatiblematrix, wrapping around a detached ligament, inserting the compositionand the ligament into a tunnel that is drilled in the bone, andattaching the ligament to the bone with sutures.

In some embodiments, a method for attachment comprises providing acomposition comprising a biocompatible matrix, wrapping around adetached tendon, inserting the composition and the tendon into the bone,injecting a PDGF solution into a tunnel that is drilled in the bone, andattaching the tendon to the bone with sutures.

In some embodiments, a method for attachment comprises providing acomposition comprising a biocompatible matrix, wrapping around adetached ligament, inserting the composition and the ligament into thebone, injecting a PDGF solution into a tunnel that is drilled in thebone, and attaching the ligament to the bone with sutures.

In some embodiments, the PDGF solution can be injected into the tunneladjacent to the implant site of the tendon wrapped with collagen matrix.In some embodiments, the PDGF solution can be injected into the medialside of the bone tunnel. In some embodiments, the PDGF solution can beinjected into the lateral side of the bone tunnel. In some embodiments,the PDGF solution can be injected at single point of the bone tunnelperimeter. In other embodiments, the PDGF solution can be injected atmultiple points of the bone tunnel perimeter. For example, the injectioncan be made at the ¼, ½, ¾, and full circumference of the tunnelopening.

In some embodiments, the concentration of the PDGF solution is about 0.1mg/ml to about 2.0 mg/ml. In some embodiments, the concentration of thePDGF solution is about 0.1 mg/ml to about 0.5 mg/ml. In someembodiments, the concentration of PDGF solution is about 0.15 mg/ml. Inother embodiments, the concentration of the PDGF solution is about 0.3mg/ml.

The present invention also provides methods for attaching a tendon intoa bone in an individual for anterior cruciate ligament reconstructioncomprising administering to said individual an effective amount of acomposition comprising a biocompatible matrix and PDGF at the interfacebetween the tendon and the bone. In some embodiments, a method foranterior cruciate ligament reconstruction comprises providing acomposition comprising a PDGF solution disposed in a biocompatiblematrix, wrapping around a long flexor tendon detached from its femoralinsertion on the lateral side, inserting the composition and the tendoninto a tunnel that is drilled obliquely though the tibia metaphysic, andattaching the long flexor tendon to the medial cortex of the tibia withsutures.

In some embodiments, a method for anterior cruciate ligamentreconstruction comprises providing a composition comprising abiocompatible matrix, wrapping around a long flexor tendon detached fromits femoral insertion on the lateral side, inserting the composition andthe tendon into a tunnel that is drilled obliquely though the tibiametaphysic, injecting a PDGF solution into the bone tunnel, andattaching the long flexor tendon to the medial cortex of the tibiawithsutures.

In some embodiments, the present invention also provides methods for theattachment or reattachment of a tendon to bone in rotator cuff injuries.Rotator cuff injuries and rotator cuff injuries treatment with an openrepair method, a mini-open repair method, and an all-arthroscopic repairmethod are discussed in WO2008/005427 and U.S. Ser. No. 11/772,646 (U.S.Publication 2008/0027470). These references are hereby incorporated byreference in their entireties. In some embodiment, a method for treatingrotator cuff injuries comprises providing a composition comprising aPDGF solution disposed in a biocompatible matrix and applying thecomposition to at least one site of tendon reattachment on the humeralhead. In some embodiments, applying the composition to at least one siteof tendon reattachment can comprise molding the composition to thecontours of the reattachment site on the humeral head. A composition,for example, can be molded into a channel formed on a surface of thehumeral head for receiving the detached tendon. The composition may beapplied to the vicinity of the insertion site of the tendon into bone tofurther strengthen the attachment.

In some embodiments, a method for treating rotator cuff tears furthercomprises disposing at least one anchoring means, such as a bone anchorin the humeral head, wherein the bone anchor further comprises a PDGFcomposition, and coupling at least one detached tendon to the boneanchor. In embodiments of the present invention, tendons can be securedto bone anchors through sutures.

In some embodiments of the present invention, the method may beperformed using arthroscopic techniques, endoscopic techniques,laparoscopic techniques, or any other suitable minimally-invasivetechniques.

PDGF solutions and biocompatible matrices suitable for use incompositions, according to embodiments of methods of the presentinvention, are consistent with those provided hereinabove.

In some embodiments, the method for attachment of a tendon or ligamentto or into a bone in an individual comprises administering to anaffected site of the individual an effective amount of a compositioncomprising: a biocompatible matrix and PDGF, wherein the biocompatiblematrix comprises pores, wherein the biocompatible matrix has a porosityof at least about 80%, and wherein at least about 50% of the PDGF isreleased within about 24 hours. In some embodiments, the PDGF is presentas a solution comprising PDGF, wherein the concentration of PDGF in thesolution is about 0.1 mg/ml to about 2.0 mg/ml. In some embodiments, theconcentration of PDGF in the solution is about 0.1 to about 0.4 mg/ml.In some embodiments, the concentration of PDGF in the solution is about0.15 mg/ml. In some embodiments, the concentration of PDGF in thesolution is about 0.3 mg/ml. In some embodiments, the biocompatiblematrix comprises a cross-linked collagen matrix. In some embodiments,the biocompatible matrix comprises a cross-linked collagen andglycosaminoglycan matrix. In some embodiments, at least about 60% of thePDGF is released within about 24 hours. In some embodiments, at leastabout 70% of the PDGF is released within about 24 hours. In someembodiments, at least about 80% of the PDGF is released within about 24hours. In some embodiments, the biocompatible matrix has a porosity ofat least about 85%. In some embodiments, the biocompatible matrix has aporosity of at least about 90%. In some embodiments, the biocompatiblematrix has a porosity of at least about 92%. In some embodiments, thebiocompatible matrix is COLLATAPE®. In some embodiments, the treatmentis of a tendon to bone attachment. In some embodiments, the treatment isof a ligament to bone attachment.

Sutures

The sutures used in the methods of the invention herein, or included inthe kits herein, may comprise PDGF. PDGF may be soaked into or coatedonto sutures by a solution comprising PDGF. In some embodiments, thePDGF is present in the solution at any concentration of about 5.0 toabout 20.0 mg/ml, for example about 7.5 to about 15 mg/ml, for example,about 10 mg/ml.

The sutures used in the methods of the invention herein, or included inthe kits herein, can be resorbable or non-resorbable in vivo. Theresorption process involves degradation and elimination of the originalmaterial through the action of body fluids, enzymes, or cells. Theresorbed sutures may be used by the host in the formation of the newtissue, or it may be other-wise re-utilized by an individual, or it maybe excreted. The sutures can be made of synthetic or natural fibers, orcombination of both.

Kits of the Invention

In another aspect, the present invention provides a kit comprising afirst container comprising a PDGF solution and a second containercomprising a biocompatible matrix.

In some embodiments, provided is a kit for treatment of a tendon orligament injury not involving a bone in an individual comprisingadministering a first container comprising a biocompatible matrix and asecond container comprising a PDGF solution, wherein the biocompatiblematrix comprises pores, wherein the biocompatible matrix has a porosityof at least about 80%, and wherein at least about 50% of the PDGF isreleased within about 24 hours.

In other embodiments, provided is a kit for attaching a tendon or aligament to a bone in an individual comprising administering a firstcontainer comprising a biocompatible matrix and a second containercomprising a platelet-derived growth factor (PDGF) solution, wherein thebiocompatible matrix comprises pores, wherein the biocompatible matrixhas a porosity of at least about 80%, and wherein at least about 50% ofthe PDGF is released within about 24 hours.

In some embodiments, the biocompatible matrix is dehydrated. In someembodiments, the solution comprises a predetermined concentration ofPDGF. The concentration of PDGF, in some embodiments, can bepredetermined according to the nature of the tendon or ligament injurybeing treated. In some embodiments, the biocompatible matrix comprises apredetermined amount according to the nature of the tendon or ligamentinjury being treated. In some embodiments, the biocompatible matrixcomprises a predetermined amount according to the nature of thetendon/ligament and the bone being treated.

In some embodiments, the biocompatible matrix comprises a cross-linkedcollagen. In some embodiments, the biocompatible matrix comprises asoluble collagen. In some embodiments, the biocompatible matrixcomprises a cross-linked collagen and a glycosaminoglycan. In someembodiments, the biocompatible matrix comprises a cross-linked collagenand chondroitin sulfate.

In some embodiments, the present invention provides a kit comprising afirst container comprising a PDGF solution, a second containercomprising a biocompatible matrix, and a syringe. A syringe, in someembodiments, can facilitate dispersion of the PDGF solution in thebiocompatible matrix for application at a surgical site, such as a siteof tendon or ligament attachment to bone. The kit may also containinstructions for use.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention in any manner.

EXAMPLES Example 1: Structural Characterization of Various CollagenMatrices

Four collagen matrices were studied to determine differences in the finestructure and porosity. The collagen matrices were obtained from KenseyNash Coporation. The matrices were made from purified collagen extractsfrom bovine dermis, which is a source primarily of Type I collagen. Thematrices are made from collagen slurries with different concentrationsof collagen, 4.5%, 5%, 6% and 7% (w/v). The dry collagen matrices (4.5%,5%, 6%, and 7%) were punched into 5 mm disks after flushing with liquidnitrogen. Disks were mounted on a stub in three different orientations(top up, bottom up, and side up), coated with gold-palladium, andexamined by scanning electron microscopy (SEM).

The SEM images revealed that there were open pores on the surface ofcollagen matrix (4.5%) and collagen matrix (5%). The SEM images revealeda dense lamina with smaller pores on the surface of collagen matrix (6%)and collagen matrix (7%). Although each of the collagen matricesappeared to be porous, based upon SEM images of cross-sectionallongitudinal slices, collagen matrix (5%) appeared to have the greatestoverall porosity as assessed by SEM. The pores in the collagen matricesappeared to not be homogeneously distributed and in some areas there areno pores at all.

The SEM images were analyzed using ImageJ software for determination ofboth pore area size and perimeter size. ImageJ is an image analysisprogram created by Wayne Rasband at the National Institutes of Health(world wide web at rsb.info.nih.gov/ij). To use the program for imageanalysis, the appropriate parameters needed to be chosen and set. Fromthe “analyze” menu, the scale from each SEM image was inputted intoprogram to set the “scale” parameter. Next, “area” and “perimeter” werechosen for the measurement parameters. Ten pores from each image wererandomly selected, measurements taken and averaged for both area sizeand perimeter size. Results for each image were recorded. Table 1 showsthe results from one analysis.

TABLE 1 Side A Cross-section Side B Area (μm²) Collagen (4.5%) 6813 ±2854  8005 ± 2135 10505 ± 4689 Collagen (5%) 11286 ± 4149  10591 ± 401611555 ± 6667 Collagen (6%) 6206 ± 2159 13643 ± 6642  4779 ± 1706Collagen (7%) 4369 ± 3183  7993 ± 3113 2342 ± 998 Perimeter (μm)Collagen (4.5%) 308 ± 65  338 ± 38 393 ± 94 Collagen (5%) 445 ± 99   417± 109  406 ± 110 Collagen (6%) 255 ± 48   428 ± 111 258 ± 50 Collagen(7%) 445 ± 77  344 ± 70 180 ± 36

Collagen matrix (5%) appeared to have the most porous structure and thepore area size was the largest from both Side A and Side B views asanalyzed by the ImageJ software.

Example 2: Cumulative PDGF Release Analysis of Various Collagen Matrices

Cumulative PDGF release from the different collagen matrices wasanalyzed. Collagen matrices were obtained from Kensey Nash Corporationand are the same as described above in Example 1. Collagen matrices (8mm disks) of each type, 5%, 6%, and 7%, were impaled on a 27½ G needle,hydrated with 80 μl of 0.3 mg/ml rhPDGF-BB and the samples wereincubated for 10 min at room temperature. The collagen disks were thenplaced in a 2 ml microtube and 2 ml elution buffer (MEM containing 2%fetal bovine serum) was added to release the rhPDGF-BB. Triplicatesamples were used for each measurement. Control samples consisted ofadding 80 μl of rhPDGF-BB to 2 ml of elution buffer. The microtubes wereshaken on an orbital shaker in a 37° C. incubator. At 10 min, 1 hr, 8hr, and 24 hr, the elutant was removed from each tube and stored at 2-8°C. An equal volume of fresh elution buffer was added to each tube. Thestored elutants of each collagen matrix were assayed for rhPDGF-BB usingthe DuoSet ELISA (R & D System) kit according to the manufacturer'sinstructions.

Collagen matrix (5%) released rhPDGF-BB with similar kinetics comparedto collagen matrix (6%) and collagen matrix (7%) (FIG. 1). The releasekinetics were characterized by an initial rapid, bolus release ofrhPDGF-BB in the first 10 minutes followed by a slower, steady releaseover the remaining 23 hour study period. Although the release kineticswere similar, the initial bolus and total amount of PDGF released fromcollagen matrix (5%) was greater than either of collagen matrix (6%) orcollagen matrix (7%).

The percent release of rhPDGF-BB from the collagen matrices was comparedto control (rhPDGF-BB in elution buffer alone). The results showed that,as above, collagen matrix (5%) had a faster and larger release of PDGFthan collagen matrix (6%) or collagen matrix (7%). (FIG. 2) The resultsof this study are also shown in Table 2.

TABLE 2 Percent PDGF Release Over Control 10 min 1 hr 8 hr 24 hrCollagen (5%) 53.5 ± 4.3 66.5 ± 4.3 70.2 ± 1.6 71.5 ± 1.5 Collagen (6%)34.1 ± 1.1 44.1 ± 0.9 48.0 ± 1.4 50.0 ± 1.3 Collagen (7%) 30.3 ± 4.639.2 ± 5.1 42.4 ± 4.9 43.5 ± 4.8

Example 3: Biopotency Study of PDGF Release from Various CollagenMatrices

The biopotency of PDGF released from the different collagen matrices wasassessed in cell proliferation assays. Samples were prepared following amodification of the protocol described above in Example 2. The elutionbuffer was changed to D-MEM containing 2% calf serum. Duplicate samplesfor each material taken at the one hour time point were used. Theconcentration of rhPDGF-BB was determined by DuoSet ELISA assay, and theresults were used as a reference for diluting the samples to aconcentration of 1 μg/ml. rhPDGF-BB at 0.3 mg/ml was used as a referencestandard and applied to all plates. Each sample was loaded into a96-well microtiter plate (black wall and clear bottom) using a startingconcentration of 1 μg/ml and then were serially diluted 1.667-foldacross the same row. Approximately 10⁴ NIH 3T3 cells were added to eachwell except for the last column on each plate, which was used as a blankcontrol. After 48 hours culture, bromodeoxyuridine (BrdU) label wasadded to each plate. After another 24 hours culture, a BrdU cellproliferation assay was conducted according to the manufacturer'sinstructions.

rhPDGF-BB released from each matrix measured in a NIH 3T3 cellproliferation assay demonstrated that the biological activity of thereleased PDGF was conserved for the three matrices analyzed. (FIG. 3).

Example 4: Study of Stability of PDGF Released from Collagen Matrices

The stability of rhPDGF released from collagen matrices was studied.Collagen matrices (8 mm disks) of each type, (5%, 6%, and 7%), wereimpaled on a 27½ G needle and hydrated with 50 μl of 1.0 mg/mlrhPDGF-BB. Each sample was incubated in a microtube filled with 0.4 mlelution buffer (20 mM sodium acetate+0.25 N sodium chloride) for 1 hr.The released PDGF was then analyzed by size exclusion HPLC. Triplicatemeasurements were taken. No significant profile shift was found for therhPDGF-BB released from collagen matrices, demonstrating the stabilityof the PDGF released from the collagen matrices.

Example 5: Study of Tenocytes Infiltration of Various Collagen Matrices

The ability of tenocytes to infiltrate into different collagen matriceswas evaluated. Collagen matrices were obtained from Kensey NashCorporation and were made from collagen slurries with collagenconcentrations of 5%, 6% and 7% (w/v) as described herein. The collagensheets (1.5-2.0 mm thick) were punched into 8 mm diameter disks for thein vitro cell migration studies.

Primary ovine tenocytes (<4 cell passages) were isolated from ovineflexor tendon. The cells were cultured in growth medium (D-MEM/P-12containing 10% fetal bovine serum (PBS)) and switched to basic medium(D-MEM/P-12 containing 2% PBS) 12 hours prior to starting the study.

50 μl (50,000 cells) of the tenocyte cell suspension in basic medium wasadded to each collagen disk sample. After 1 hr incubation at 37° C. and5% CO₂ atmosphere without medium emersion, the cell-seeded disks weretransferred to a 24-well plate pre-filled with 2 ml of basic mediumalone or in combination with rhPDGF-BB (30 ng/ml). After 12 h of staticculture, the plates with cell-seeded disks were placed on an orbitalshaker (60 rpm) in the incubator. Medium changes were provided every 48h. On day 6, quadruplicate samples from each collagen matrix andtreatment were taken for histological assessment.

Generally, the histological assessment was similar to the followingdescription. After six days' culture, the cell culture media was removedfrom each well and replaced with 4% phosphate buffered formalin (PBF).The samples were fixed in PBF for approximately 30 minutes at roomtemperature (RT). The samples were placed under vacuum at RT for aperiod of no less than 1 hour to complete cellular fixation. Using ashaker platform and a vacuum chamber, the specimens were then dehydratedover a period of approximately 5.5 hours through an increasing series ofethanol concentrations (70%-80%-95%-100%) at RT, cleared in 100% xyleneover a period of 2 hours at RT, and infiltrated in paraffin wax for aperiod of no less than 2 hours. Samples from each treatment group andmatrix were then embedded. The embedded specimens were removed fromtheir embedding moulds, “trimmed” and “faced” with a rotary microtome toexpose outermost surface of all specimens, and then placed in thefreezer overnight. Using a rotary microtome, a warmed water bath, andpre-labeled glass microscope slides, sections (2 sections per slide)were taken at 4-6 microns at 3-4 levels approximately 100-150 micronsapart. Slides were dried overnight in an oven at 60° C. Finally slideswere stained with Hoescht fluorescence stain and viewed.

The results showed that tenocytes infiltrated into collagen matrix (5%)with PDGF, while tenocytes did not appeared to infiltrate into collagenmatrix (6%) and collagen matrix (7%). For collagen matrix (6%) andcollagen matrix (7%) samples most of the tenocytes accumulated at theedge of the disks. Therefore it appeared that the greater porosity ofcollagen matrix (5%) allowed for a higher number of infiltrating cellsinto the collagen matrix.

Example 6: Treatment of Achilles Tendon Injury with Collagen/PDGFCompositions

An exemplary study, as described below, was used to evaluate theefficacy of a composition and methods in accordance with the presentinvention. The present invention includes compositions, and methods ofuse thereof, that comprise an rhPDGF-BB solution mixed with a collagenmatrix. For example, the collagen matrix is a flowable collagen matrixand comprises a cross-linked bovine tendon collagen andglycosaminoglycan (GAG) matrix. Flowable compositions are readilyprovided to the injury site by a syringe or other suitable means.

Sheep, or other suitable test subjects, were used in this exemplarystudy as a model for human Achilles tendon repair. Sheep areparticularly suitable due to the similarity of the sheep Achilles tendonsize to the human Achilles tendon. Additionally, sheep are of sufficientsize to allow for standard orthopedic techniques and placement of thecomposition. The sheep used in this study were skeletally mature (asdetermined by age [3.5 years and older] and dental wear) with normalambulation, and weighed at least 120 lbs and acclimated at the time ofsurgery. Sheep were fed and watered in accordance with a standard smallruminant diet. Food and water were withheld for appropriate studyrelated events such as anesthesia.

The study used three treatment groups (n=8/group) in which the followingtest compositions were provided and applied to the injury site of acuteAchilles tendon transection: (1) flowable collagen matrix in buffer, (2)flowable collagen matrix in buffer with 0.3 mg/mL (150 μg) rhPDGF-BB,and (3) flowable collagen matrix in buffer with 1.0 mg/mL (500 μg)rhPDGF-BB. Other suitable treatment groups are used such as, forexample, those that use compositions with other suitable concentrationsof rhPDGF-BB.

An exemplary flowable collagen matrix is the Integra™ Flowable WoundMatrix (IFWM) of Integra LifeSciences Corporation, Plainsboro, N.J.Other suitable matrices known in the art can also be used.

Experimental Overview

The Achilles tendons of sheep enrolled into this study were transected,followed by immediate repair. The sheep were divided into threeexemplary test groups (n=8/group) in which the following compositionswere used: 1) flowable collagen matrix in 20 mM sodium acetate buffer(pH 6.0+/−0.5), placed at the re-approximated tendon ends (stabilizedwith a modified Mason Allen suture design) (control), 2) flowablecollagen matrix, in 20 mM sodium acetate buffer containing 0.3 mg/mlrhPDGF-BB, placed at the re-approximated tendon ends (stabilized with amodified Mason Allen suture) and 3) flowable collagen matrix in 20 mMsodium acetate buffer containing 1.0 mg/ml rhPDGF-BB, placed at there-approximated tendon ends (stabilized with a modified Mason Allensuture). Suturing material was used, such as #1 Ethilon nylon suture(Ethicon Endo-Surgery, Inc, Cincinnati, Ohio). Biomechanical performanceand histological response of asimulated Achilles rupture andreattachment were determined. The treatment allocations and number ofanimals for both the biomechanical performance and the histologicalresponse tests are outlined in Table 3.

TABLE 3 Treatment allocations Treatment Animals Group (n) rhPDGF-BBEndpoint 1 Collagen 8 0 Biomechanics Flowable Wound (N = 6)/Matrix(IFWM) + Histology Buffer (N = 2) 2 IFWM + 8 0.3 mg/mlBiomechanics 0.3 mg/ml (N = 6)/ rhPDGF-BB Histology (N = 2) 3 IFWM + 81.0 mg/mL Biomechanics 1.0 mg/ml (N = 6)/ rhPDGF-BB Histology (N = 2)Contralateral Achilles tendons from treatment group 1 will also becollected for biomechanics (n = 6) and histology (n = 2)

Following placement of the test materials, the incision was closed usingstandard surgical techniques and a splint placed on the lower foot toprevent knuckling during ambulation. During the week post-surgery, thesurgical site was monitored for abnormal healing or wound dehiscence.The animal was allowed to ambulate normally for 8 weeks and hadultrasound evaluations performed at 2, 4 and 8 weeks post-operative.Eight weeks post-surgical, all animals were euthanized and the Achillestendon was collected, including the proximal and distal musculotendinousjunctions, for histological and biomechanical assessment. Normal,unoperated tendons, and their musculotendinous junctions, were collectedfrom the contralateral hind limb of control animals (i.e., flowablecollagen wound matrix alone) so that histologic and biomechanicaltesting may be performed for normal, untreated tendons. The skin fromthe initial surgical site and contralateral controls are also taken forhistological evaluation.

Surgical Protocol

On the day of surgery, an IV injection consisting of Diazepam (0.22mg/kg) and Ketamine (10 mg/kg) was given for induction of generalanesthesia. A cuffed endotracheal tube was placed and general anesthesiawas maintained with isofluorane (1.5% to 3.0%) in 100% oxygen (2 L/min)through a rebreathing system. The animal was placed on a ventilator toassist respiration. A stomach tube could be placed.

With the animal in left lateral recumbency, the wool was removed fromthe right leg to provide adequate access to prepare the site forsurgery. The skin over the right ankle joint was prepared for asepticsurgery using alternating scrubs of povidone-iodine (Betadine) andalcohol. The surgical site was then draped for aseptic surgery. Anincision was made over the lateral aspect of the leg and then deepenedthrough the subcutaneous tissues to expose the tendon at its insertionsite on the calcaneus. The larger branch of the gastrocnemius tendon wasthen isolated. The Achilles tendon insertion to the calcaneus waspalpated and marks were placed at 2, 4, and 6 cm from the insertion sitewith a sterile marker.

Prior to surgery, the matrix and appropriate buffer solution (20 mMsodium acetate buffer, pH 6.0+/−0.5)+/−rhPDGF-BB, were combined byadding 6.0 cc of the buffer+/−rhPDGF-BB, to 3.0 cc of the flowablecollagen material (IFWM) (2:1 v/v), using the supplied, labeled sterilesyringes containing the dry collagen and buffer+/−rhPDGF-BB. Thematerials were combined by removing the tip cap from the syringecontaining the rhPDGF-BB and replacing it with the supplied, sterileluer lock connector to which the syringe containing the dry collagen wasthen connected. Hydration of the collagen matrix was accomplished bydispensing all buffer+/−rhPDGF-BB into the collagen syringe and mixingby depressing the plungers back and forth at least 15 times until themixture appears homogeneous and all the material can be moved back andforth easily between syringes. The hydrated flowable collagen matrix wasaliquoted into 0.5 cc volumes in sterile containers and stored at 4° C.until use.

After exposure of the tendon, a stabilizing suture (#1 Ethilon) wasplaced using a modified Mason Allen design with one end at the 2 cm markand one end at the 6 cm mark. The Mason Allen suture was kept taut tomaintain the tendon length, but one end was left untied for themanipulation of the tendon ends. The tendon was then transected at orabout the 4 cm mark, after which the tendon ends were re-approximatedand the free end of the Mason Allen suture was tied at the distal end ofthe tendon. A photograph was taken of the transected tendon with a rulerto document the distance from the calcaneal insertion. See, e.g., theexemplary sutured, transected tendon as depicted in FIG. 4. A small tailin the suture was left at the distal end and a small identifying suture(3-0) is placed at the proximal end for identification after necropsy.The overlying soft tissue was closed, leaving a space around the tendontransection for insertion of the flowable matrix. The hydrated flowablecollagen matrix material was then placed at the tendon ends in thefollowing manner: The 0.5 cc sterile aliquot was expressed from thesyringe through a 20 gauge needle, with the beveled side facing towardsthe transected tendon ends. Half (0.25 cc) of the sterile aliquot wasexpressed along the surface of each of the transected tendon ends. Carewas taken to ensure that the material was evenly distributed in the gapspace. See, e.g., an exemplary flowable composition of the presentinvention being delivered the exemplary sutured, transected tendon asdepicted in FIG. 5.

The overlying subcutaneous tissue and skin were closed according tostandard surgical procedure. A splint made of OrthoGlass was formed onthe lower foot to prevent knuckling during ambulation. Immediately aftersurgery, the sheep were transferred from the operating table andobserved until the swallowing reflex returns, at which point they wereextubated. Upon completion of surgery, the sheep were propped in sternalrecumbency, and then housed for the duration of the study. Splintingand/or casting of the sheep were used to reduce knuckling and/oroverstretching of the repaired tendon. Postoperative analgesia wereprovided, and the animals were managed according to standardpost-operative care procedures.

Clinical Observations

Animals were observed twice daily until sacrifice. During the firstweek, the surgical site was observed, and photos obtained, for abnormalhealing and wound dehiscence. During the entire post-surgical studyperiod, the animals were observed for general attitude, appetite,operated limb use (e.g., lameness), and appearance of the surgical site.Antibiotics were administered to an animal when infection developed atthe surgical site, and noted in the observations. Ultrasound images, forboth the operated and the contralateral, unoperated, Achilles tendonwere obtained for all treatment sites 2-, 4-, and 8-weeks post-surgicalusing methods known in the art. Eight (8) weeks following the indexsurgery, all animals were sacrificed for tissue harvest.

Biomechanical Testing

Mechanical performance of a stimulated Achilles tendon rupture andreattachment using three different treatment groups as discussed aboveunder the Experiment Overview Section was determined.

Materials and Methods

Following harvest, specimens were wrapped in saline soaked gauze andstored at −20° C. until testing. The metatarsus was potted in 2″ PVCpipe using high strength polymethylmethacrylate (PMMA). Specimens werekept moist during the potting preparation and biomechanical testing witha saline spray at 15 minute intervals. The potted metatarsus was mountedin a custom-designed testing fixture that was rigidly attached to thematerials testing system loading frame (MTS MiniBionix II, Edan Prairie,Minn.). A custom-designed cryoclamp was implemented to preserve thenatural cross section of the Achilles tendon and minimize soft tissueslippage and applied a uniaxial traction force to the construct at anangle of approximately 1350 to the potted metatarsus. This was done tomimic the physiological force vector of the tendon. Testing commencedwhen a thermocouple attached to the cryoclamp registered −22° C., atemperature previously reported to be sufficient to ensure securecoupling between the tendon and clamps. No tissue-embedded suturematerial was cut prior to testing. Thus, the biomechanical resultsrepresent the combined mechanical contribution of the embedded sutureand the reparative tissue.

Four retroreflective markers were sutured or glued on to the pottedconstruct: one on the calcaneus immediately adjacent to the repair site,two on the tendon—one proximal and one distal to the Achillestendon-repair tissue interface. The fourth was glued on the cryoclamp.Three cameras (Motion Analysis, Santa Rosa, Calif.) recorded the spatialmovement of the markers at 60 Hz. Marker displacement measurements usingthe camera system allowed for real-time monitoring of local tissuedisplacement/strain within the reparative tissue.

Phase 1: 30 Cycle Dynamic Preconditioning

A cyclic loading test was initially employed to pre-condition therepaired tendon. Using a force control protocol, a 10 Newton (N) preloadwas applied to the construct for two minutes. This was designated theinitial configuration for all constructs. The repaired constructs werethen cyclically preconditioned in a force-control protocol from 10 to 50Nat 0.25 Hz for 60 cycles to reach a steady-state. Sixty (n=60) cycleswas chosen based on previous experiments in our laboratory that havedemonstrated that the slope of the displacement versus time curveconverges between 50 and 60 cycles. Parameters of interest includedconditioning elongation, defined as the difference in peak-to-peakdisplacement between the first and sixtieth cycles, and peak-to-peakelongation, defined as the average of the difference between the localminimum and maximum of the 58th, 59th, and 60^(th) cycles.

Quasi-Static Failure Loading

Following preconditioning, the repaired constructs were loaded tofailure under displacement control at a rate of 1 mm/s. Biomechanicalparameters of interest included ultimate load-to-failure, quasi-staticglobal construct stiffness (defined as the slope of theload-displacement curve), local repair tissue stiffness, elongation atfailure, and energy absorbed at failure. Finally, the failure mechanismwas documented for each specimen. Digital images and/or video were takenprior to testing, during the ramp to failure procedure, and followingconstruct failure.

Statistical Analysis

A One-Way ANOVA followed by a Tukey's post hoc multiple comparison testwas used to identify significant differences in continuous biomechanicalparameters between the IFWM control and the 0.3 mg/mL PDGF and 1.0 mg/mLPDGF treatment groups. Significance was set at p≤0.05 and all analyseswere performed with SigmaStat 3.1 (Systat Software, Inc., San Jose,Calif.).

Results

No gross visual differences were identified between the three treatmentgroups. Two of the six (33%) constructs in the IFWM+0.3 mg/ml rhPDGF-BBgroup displayed an evident hematoma on the lateral aspect of the repairtissue. The presence of this hematoma affected the biomechanicalproperties of the repair, evidenced by failure initiating at the exactlocation of the hematoma at unusually low loads. Data analysis wasperformed with (n=6) and without (n=4) these two hematoma affectedspecimens. Raw data from the cyclic preconditioning component of testingincluding the two hematoma affected specimens in the 0.3 mg/mlrhPDGF-BBgroup are presented in Table 4. No significant differences inconditioning elongation (p=0.636) or peak-to-peak elongation (p=0.813)were identified between the IFWM, IFWM+0.3 mg/ml rhPDGF-BB, or IFWM+1.0mg/ml rhPDGF-BB treatment groups (FIG. 6A).

TABLE 4A Summary Data from Cyclic Preconditioning Analysis. DataReported as Mean ± S.E.M. Conditioning Peak-to-Peak ElongationElongation Treatment n (mm) (mm) IFWM 6 0.908 ± 0.153 1.041 ± 0.103IFWM + 0.3 6 0.905 ± 0.110 1.084 ± 0.054 mg/mL rhPDGF-BB IFWM + 1.0 60.754 ± 0.119 1.018 ± 0.051 mg/mL rhPDGF-BB iCTL 6 0.188 ± 0.045 0.553 ±0.016

Similarly, no significant differences in conditioning elongation(p=0.709) or peak-to-peak elongation (p=0.947) were identified betweenthe IFWM, IFWM+0.3 mg/ml rhPDGF-BB, or IFWM+1.0 mg/ml rhPDGF-BBtreatment groups. FIG. 6B. Raw data from the cyclic preconditioningcomponent of testing with hematoma affected specimens excluded from theanalysis are presented in Table 4B.

TABLE 4B Summary Data from Cyclic Preconditioning Analysis. DataReported as Mean ± S.E.M. Conditioning Peak-to-Peak ElongationElongation Treatment n (mm) (mm) IFWM 6 0.908 ± 0.153 1.041 ± 0.103IFWM + 0.3 4 0.829 ± 0.123 1.084 ± 0.049 mg/mL rhPDGF-BB IFWM + 1.0 60.754 ± 0.119 1.018 ± 0.051 mg/mL rhPDGF-BB iCTL 6 0.188 ± 0.045 0.553 ±0.016 ** two of the six specimens in this group displayed an evidenthematoma on the lateral aspect of the repair tissue at which tissuerupture clearly initiated during RTF testing. These have been removedfrom statistical comparison.

Raw data from the ramp to failure component of the biomechanical testingwith the two hematoma affected specimens included in the analysis arepresented in Table 5A. No significant differences in the surgicallyrepaired Achilles tendons were identified for any quasi-static parameterbetween the IFWM, IFWM+0.3 mg/ml rhPDGF-BB, or IFWM+1.0 mg/ml rhPDGF-BBtreatment groups (p>0.05, Table 5, FIG. 7A). Though not significant, the1.0 mg/mL rhPDGF-BB dose resulted, on average, in a 57.4% and 55.0%increase in ultimate force to failure relative to the IFWM control and0.3 mg/mL rhPDGF-BB groups, respectively. No significant difference inenergy absorbed at failure was identified between the three treatmentgroups (p=0.209: IFWM, 6423.33±1811.26 N*mm; 0.3 mg/mL rhPDGF-BB,7346.00±2989.94 N*mm; 1.0 mg/mL PDGF, 12173.33±2049.62 N*mm), though theenergy absorbed at failure in the 1.0 mg/mL rhPDGF-BB treatment groupwas, on average, 89.5% and 65.7% greater than the IFWM control and 0.3mg/mL rhPDGF-BB treatment groups, respectively.

TABLE 5A Summary Data from Ramp to Failure Analysis. Data Reported asMean ± S.E.M. Global Construct Global Displace- Ultimate Load Stiffnessment at Failure Treatment n (N) (N/mm) (mm) IFWM 6  922.04 ± 149.9196.61 ± 8.56 14.55 ± 2.42 IFWM + 0.3 6  936.50 ± 231.74 82.43 ± 9.7514.66 ± 2.04 mg/mL rhPDGF-BB IFWM + 1.0 6 1451.71 ± 125.08 113.11 ±7.14  18.02 ± 1.24 mg/mL rhPDGF-BB iCTL 6 3767.54 ± 224.43 176.97 ±10.75 27.04 ± 1.95

Raw data from the ramp to failure component of the biomechanical testingwith the two hematoma affected specimens excluded from the analysis arepresented in Table 5B. No significant differences in the surgicallyrepaired Achilles tendons were identified for any global quasi-staticparameter between the IFWM, IFWM+0.3 mg/ml rhPDGF-BB, or IFWM+1.0 mg/mlrhPDGF-BB groups (p>0.05, Table 5B, FIG. 7B). Though not significant,the 1.0 mg/ml PDGF dose resulted, on average, in a 57.4% and 22.2%increase in ultimate force to failure relative to the IFWM control and0.3 mg/ml PDGF groups, respectively. No significant difference in energyabsorbed at failure was identified between the three groups (p=0.247:IFWM, 6423.33±1811.26 N*mm; 0.3 mg/ml PDGF, 9850.75±4006.36 N*mm; 1.0mg/ml PDGF, 12173.33±2049.62 N*mm), though the energy absorbed atfailure in the 1.0 mg/ml PDGF treatment group was, on average, 89.5% and23.6% greater than the IFWM control and 0.3 mg/ml PDGF treatment groups,respectively.

TABLE 5B Summary Data from Ramp to Failure Analysis. Data Reported asMean ± S.E.M. Global Construct Global Displace- Ultimate Load Stiffnessment at Failure Treatment n (N) (N/mm) (mm) IFWM 6  922.04 ± 149.9196.61 ± 8.56 14.55 ± 2.42 IFWM + 0.3  4** 1188.31 ± 266.12 93.81 ± 7.1716.16 ± 2.61 mg/mL rhPDGF-BB IFWM + 1.0 6 1451.71 ± 125.08 113.11 ±7.14  18.02 ± 1.24 mg/mL rhPDGF-BB iCTL 6 3767.54 ± 224.43 176.97 ±10.75 27.04 ± 1.95

Including the two hematoma affected specimens in the analysis (Table 6,FIG. 8 (left)), local repair tissue stiffness in the 1.0 mg/mL rhPDGF-BBtreatment group (n=6, 312.56±20.86 N/mm) was, on average, 77.3% greaterthan the local stiffness quantified in the 0.3 mg/mL treatment group(n=6, 176.30±31.17 N/mm). This difference was statistically significant(p=0.012). No significant difference in local repair tissue stiffnesswas identified between the 1.0 mg/mL PDGF treatment group and the IFWMcontrol (n=6, 215.23±33.23 N/mm, p=0.075). With the two hematomaaffected specimens excluded from the analysis (FIG. 8(right)), localrepair tissue stiffness in the 1.0 mg/ml rhPDGF-BB treatment group was,on average, 39.7% greater than the local stiffness quantified in the 0.3mg/ml treatment group (n=4, 223.72±11.46 N/mm). This difference was notstatistically significant (p=0.096). However, local repair tissuestiffness in the 1.0 mg/ml group was significantly greater (45.2%,p=0.039) than that of the IFWM control (215.23±33.23 N/mm).

TABLE 6 Summary Data from Ramp to Failure Analysis. Data Reported asMean ± S.E.M. Local Construct Local Construct Stiffness StiffnessTreatment n (N/mm) n (N/mm) IFWM 6 215.23 ± 33.23 6 215.23 ± 33.23IFWM + 0.3 6 176.30 ± 31.17  4** 223.72 ± 11.46 mg/mL rhPDGF-BB IFWM +1.0 6 312.56 ± 20.86 6 312.56 ± 20.86 mg/mL rhPDGF-BB iCTL 6  882.87 ±124.03 6  882.87 ± 124.03

Of the eighteen (n=8) treatment constructs tested, n=16 (88.9%) failedat the proximal interface between the preparative tissue and intactAchilles. Of note, two of the sic constructs in the IFWM+0.3 mg/mLrhPDGF-BB treatment group displayed a hematoma on the lateral aspect ofthe repair tissue that was grossly visible during dissection andsubsequent biomechanical testing. In both constructs, failure initiatedat these regions. The remaining n=2 (11.1%) treatment constructs failedat the distal interface between the reparative tissue and the intactAchilles. Mode of failure for the n=6 intact contralateral Achillestendon constructs varied. Two (n=2, 33.3%) intact constructs failed viametatarsal fracture within the PMMA potting material. Three (n=3, 50%)intact constructs failed via mid-substance tearing of the Achillestendon, and one (n=1, 16.7%) failed via calcaneal avulsion.

Conclusion

After eight weeks in vivo, the biomechanical data observed for the grouptreated with IFWM+1.0 mg/mL rhPDGF-BB were consistently increased and onaverage exhibited a greater healing response compared to the IFWMcontrols and IFWM+0.3 mg/mL rhPDGF-BB treatment groups. This dosingeffect was manifested as a 55%/22.2% (n=6/n=4) and 57.4% increase inultimate load-to-failure relative to the 0.3 mg/mL and IFWM alonetreatment groups, respectively. Repair (i.e., local) tissue stiffnesswas increased on average by 77.3%/39.7% (n=6/n=4) and 45.2% relative tothe 0.3 mg/mL rhPDGF-BB and IFWM alone treatment groups, respectively.Additionally, the ultimate force observed in this study for the IFWM+1.0mg/mL rhPDGF-BB group was increased compared to other studies whichutilized a matrix (34.9-fold higher than a 24 week repair using acollagen patch combined with a platelet rich plasma fibrin matrix(Sarrafian et al., Trans ORS, 33:322 (2008)) or a protein (1.9-foldhigher than 3 week repair treated with CDMP-2 (Virchenko, Arch OrthopTrauma Surg, 128:1001-1006 (2008)).

Histological Testing

Tissue Harvest and Trimming

Following euthanasia, operated Achilles tendons were harvested, splintedto prevent curling of the tissue, and placed in 10% neutral bufferedformalin for histological processing. In addition to the operatedAchilles, two unoperated contralateral Achilles tendons were harvestedfor histological analysis. After fixation for 24 hours, each Achillestendon was bisected into medial and lateral halves using a scalpel. Theproximal end of the Achilles was notched to preserve orientationthroughout the histological processing. One animal in the 1.0 mg/mlrhPDGF-BB group was euthanized after 40 days (5.8 weeks) of healing dueto reasons unrelated to the study (pneumonia).

Histological Processing

All specimens (n=8) were further fixed, dehydrated, cleared,infiltrated, and embedded using standard paraffin histology techniquesand equipment (Shandon Citadel 2000 Processor and Shandon Histocentre 2,Thermo Shandon, Inc, Pittsburgh, Pa.). The paraffin blocks were facedand approximately 10 micron sections cut on Shandon Finesse rotarymicrotome (Thermo Shandon, Inc, Pittsburgh, Pa.). Five sections were cutper specimen, for a total of 40 sections. Each histological section wasstained with Hematoxylin-Eosin (H&E). High-resolution digital imageswere acquired field by field for the entire stained slide includingregions of interest using an Image Pro Imaging system (MediaCybernetics, Silver Spring, Md.) and a Nikon E800 microscope (AG Heinze,Lake Forest, Calif.).

Qualitative Histopathology

All tissue sections were evaluated to assess the quality of thereparative/healing tissue, the native tendon/reparative tissueinterface, vascularization, inflammation, and collagen density/fiberorientation. Sections were assessed blinded to treatment. Tendonretraction was also measured via calibrated gross digital images usingImage Pro Plus imaging system.

Results

Tendon Retraction

All operated specimens displayed some degree of tendon retraction after8 weeks of healing. On average, the Achilles tendon retracted 55.7±16.1mm for the No Dose group, 39.8±4.5 mm for the 0.3 mg/ml rhPDGF-BB dosegroup, and 44.4±1.5 mm for the 1.0 mg/ml rhPDGF-BB dose group.

Histopathology

After 8 weeks of healing, the flowable collagen was visible in all ofthe specimens regardless of the rhPDGF-BB dose. The flowable collagenwas generally located more towards the distal end of the repair site. Ina few cases the flowable collagen had migrated out of the center of therepair site towards the dorsal surface of the reparative tissue. In onespecimen the flowable collagen was observed dispersed over the entirelength of the repair site. Only mild inflammation was observed in andaround the flowable collagen. Inflammation was judged to be slightlyincreased in the flowable collagen+rhPDGF-BB groups (0.3 mg/ml and 1.0mg/ml doses) compared to No Dose specimens. Inflammation generallyconsisted of multinucleated foreign body giant cells and/or mononuclearinflammation (lymphocytes, monocytes and plasma cells). Neutrophils weregenerally not observed. Fibroplasia with new collagen production byactivated fibroblasts was observed within the flowable collagenregardless of the rhPDGF-BB dose. Although some fibroplasias wasobserved within the flowable collagen, there was a discontinuity withinthe reparative collagen fibers. The proximal and distal ends of thenative Achilles tendon were generally well integrated with the newcollagen fibers of the reparative tissue. In a few cases, the distalinterface demonstrated slightly better integration as compared to theproximal interface. The proximal interface contained many youngfibroblasts producing immature collagen with a distorted native Achillestendon end combined with mild inflammation.

There was relatively high fibroplasia and collagen production byactivated fibroblasts with a high density of collagen fibers regardlessof treatment or rhPDGF-BB dose. Primary collagen fiber alignment wascoincident with the native Achilles collagen fiber direction, asobserved using polarized light. Fibroblast density was similar betweentreatments. In one specimen, the density of fibroblasts was relativelylower and the collagen fibers were more immature, less dense, and lessoriented. The collagen fibers were generally aligned parallel to thenative Achilles fiber direction although no differences were observedbetween groups for collagen or fibroblast density.

Inflammation was observed within the repair sites for all treatments andwas generally mild. Inflammation consisted of multinucleated foreignbody giant cells with mononuclear inflammation (lymphocytes, monocytesand plasma cells). Neutrophils were generally not observed. Inflammationwas associated with the presence of suture material and flowablecollagen, and most probably due to localized damage of the host tissue.Abundant vascularization was also observed within the repair sites forall treatments. Increased inflammation was associated with more abundantvascularization. Vascular hypertrophy, reactive vessels with plumpendothelial cells and thickening of vessel walls, was observed for allrhPDGF-BB doses. There were more hypertrophic vessels noted in thespecimens treated with the 0.3 mg/ml and 1.0 mg/ml doses as compared tothe No Dose specimens. These vessels were usually located within thereparative tissue in superficial regions of the dorsal surface. In onespecimen (40 days post operation), there was abundant vascularization,but these vessels were smaller and hypertrophic vessels were notobserved. Hemorrhage, found in conjunction with a hematomatomatous spaceand fibrin, was observed in 2 specimens.

Conclusion

In summary, based on the limited number of samples tested, there were noqualitative differences in Achilles tendon repair between the 0.3 mg/mldose, 1.0 mg/ml dose, and control (No Dose) groups. The presence ofrhPDGF-BB did not prevent healing nor did it illicit a negative responsehistologically. There was similar healing between the 0.3 mg/ml dose and1.0 mg/ml dose treatment groups. All operated specimens displayed somedegree of tendon retraction. The flowable collagen was visible in allspecimens regardless of rhPDGF-BB treatment or dose, and was generallylocated towards the distal end of the repair site. Inflammation wasgenerally mild and considered of multinucleated foreign body giant cellswith mononuclear inflammation (lymphocytes, monocytes and plasma cells).Inflammation was judged to be slightly increased within the flowablecollagen treated with rhPDGF-BB (0.3 mg/ml and 1.0 mg/ml dose) ascompared to the No Dose group. In the reparative tissue there wassubstantial, ongoing fibroplasias with a high density of collagen fibersthat was irregardless of rhPDGF-BB treatment or dose. Reparativecollagen fiber alignment was generally paralleled to the native Achillescollagen fiber direction. Collagen and fibroblast density was similarbetween treatments. Abundant vascularity was observed in all specimens.There were judged to be more hypertrophic vessels noted in the specimenswith the 0.3 mg/ml and 1.0 mg/ml doses as compared to the No Dose group.In a few cases, the distal interface displayed slightly betterintegration as compared to the proximal interface, where there was lessmature collagen and a distorted native Achilles tendon end combined withmild inflammation. This may be the result of continuous tendonretraction and concomitant local tissue damage caused by the sutureslippage.

Example 7: Normal and Diseased Primary Human Tenocytes Proliferate inResponse to rhPDGF-BB

This study determined whether rhPDGF-BB directly activated proliferationand/or chemotaxis of primary tenocytes derived from patients withtendinopathies. Such findings can support the notion of therapeuticpotential of rhPDGF-BB in tendinopathies.

Patients and Methods

Patients

Ten patients with tendinopathies were involved in this study, includingfive patients with Achilles tendinopathy and five patients withtendinopathy of the posterior tibial tendon (PTT). Additional fivepatients were involved who underwent full joint replacement of the knee.

Primary Cultures of Tenocytes

Tendon tissue which would otherwise be discarded was obtained fromnormal and injured tendons during reconstructive surgery proceduresperformed for clinical indications. These tissues included thetendinopathic portion of the Achilles or PTT tendons, as well as thehealthy (non-tendinopathic) portion flexor digitorum longus (FDL) tendontissue, Achilles tendon tissue, and Patellar tendon tissue. Primarytenocyte explant cultures were obtained from these tissues and tested atpassages 3 to 5. Tenocyte identity was confirmed by assessing theexpression of a tenocyte-specific gene scleraxis and genes for collagensa1(I), a2(I), and a1(III) in real-time PCR assays with specific primers.

Cell Proliferation

Tenocyte monolayers were trypsinized, resuspended in DMEM/F12 mediumcontaining 0.5% dialyzed fetal bovine serum, allowed to attachovernight, and then incubated with titrated concentrations rhPDGF-BB for24 h. Changes in cell proliferation rates were assessed based on BrdUincorporation during DNA synthesis in cells using a commerciallyavailable assay (Roche Applied Science, Indianapolis, Ind.). Eachculture was tested in triplicates for each dose of rhPDGF-BB.

Cell Migration

Tenocyte monolayers were trypsinized, resuspended in DMEM/F12 mediumcontaining 0.5% dialyzed fetal bovine serum and placed in the upperchamber of the 96-well ChemoTx® disposable cell migration system (NeuroProbe, Gaithersburg, Md.). The lower chambers contained titratedconcentrations of rhPDGF-BB. Tenocytes were allowed to migrate acrossthe membrane separating the chambers for 48 h. 96-well plates were thenspun down and freeze thawed three times to lyse the migrated cells. Theamount of viable migrated cells was measured based on cytoplasmiclactate dehydrogenase (LDH) using a commercially available kit fromPromega (Madison, Wis.).

Statistical Analysis

One-way ANOVA was used to determine whether stimulation with rhPDGF-BBaffects tenocyte proliferation in a dose-dependent fashion.

Results

Only tenocyte cultures but not control pulmonary fibroblast cultures orcontrol primary T lymphocyte cultures expressed scleraxis mRNA, whereastenocytes and fibroblasts but not lymphocytes expressed the collagengene mRNAs.

In all cases, tenocytes from tendon tissues involved or not involved inthe disease process responded to rhPDGF-BB stimulation by acceleratingBrdU incorporation (p<0.05, one-way ANOVA). The responses weredose-dependent and were observed at 10, 50 and 150 ng/mL of rhPDGF-BB.Even though all cell cultures responded to rhPDGF-BB stimulation, therewas significant variability among patients in the magnitude of BrdUincorporation after rhPDGF-BB stimulation. Incorporation of BrdUincreased from a minimum of 2.1±0.2 fold to a maximum of 10.7±0.5 foldcompared to control non-stimulated cultures. Tenocytes from fivepatients responded paradoxically, with a greater increase in BrdUincorporation at a lower (10 ng/mL) than higher (50 and 150 ng/mL)concentrations of rhPDGF-BB. Such paradoxic response was observed intenocytes derived from both tendinopathic and normal tissues of thesepatients. Tenocytes derived from healthy tendons of four patientsincorporated twice more BrdU in response to rhPDGF-BB stimulation thandid tenocytes derived from the diseased tissues. In one patient,tenocytes from the diseased tissue incorporated four fold more BrdU inresponse to rhPDGF stimulation than did tenocytes from the uninvolvedtissue.

In all cases, tenocytes were chemotactically responsive to rhPDGF-BB at50 ng/mL and 150 ng/mL. Tenocytes were not exposed to 10 ng/mL rhPDGF-BBfor chemotaxis experiments because of low response in pilot experiments.Again, responses were dose-dependent, with greater chemotaxis to 150ng/mL than to 50 ng/mL of rhPDGF-BB. However, tenocytes from 5 patientsresponded with greater chemotaxis to 50 ng/mL than to 150 ng/mL ofrhPDGF-BB, with significant decline in the number of migrated cell(p<0.05, two-sided Student's t-test). There was variability amongpatients in the maximal chemotactic response to rhPDGF-BB, from 1.4±0.1to 4.0±0.5 fold increase compared to non-stimulated control. There wasno statistically significant difference (p>0.05) in tenocyte chemotaxisto rhPDGF-BB within matching tenocyte cultures derived fromtendinopathic or from healthy tendon tissues.

Conclusion

The results of these experiments suggest that tenocytes derived fromhealthy and tendinopathic tissues respond to rhPDGF-BB by increasingproliferation and chemotaxis rates. Importantly, tenocytes from somepatients showed paradoxical response to PDGF, in which higher dosescaused less effect than lower doses. Equally important, tenocytes fromdiseased tendons were in some cases differentially responsive to PDGFversus tenocytes from healthy tendons, implying that proper dosing maybe of paramount importance in the clinical setting.

Example 8: Evaluation of Four Collagen Matrices in Combination withRecombinant Human Platelet-Derived Growth Factor-BB (rhPDGF-BB) forApplication in Treatment of Tendon to Bone Reattachment

This study assessed the physical characteristics, biocompatibility (invitro and in vivo) and stability (biodegradation) of four collagenmatrices that could be used in conjunction with rhPDGF-BB at thetendon-bone interface for application in the treatment of tendon to boneattachment, such as anterior cruciate ligament reconstruction.

Methods

(i) Materials and Preparations

Four Type I collagen matrices (pads) were evaluated in this study. Threefibrous collagen matrices (A, Band C) were bovine dermal derivatives ofdiffering collagen densities (A=4.5% collagen; B=5% collagen, C=6%collagen; BIOBLANKET™ collagen made from different percentage ofcollagen slurry provided by Kensey Nash Corporation) and the fourthfibrous matrix (D) was a bovine tendon derivative characterized as“highly porous” (90% porosity) (COLLATAPE®, Integra LifeSciences). Allcollagen sheets (1.5-2.0 mm thick) were punched to 8 mm diameter discsfor physical characterization and in vitro (cytocompatibility)evaluation. In vivo (biocompatibility and biodegradation) evaluationswere performed utilizing 1×1 cm² pads. rhPDGF-BB: recombinant humanplatelet derived growth factor (rhPDGF-BB) at 0.3 mg/ml was alsoprepared.

Cell source is primary ovine tenocytes (<4 passages) isolated from ovineflexor tendon that were cultured in growth medium (DMEM/F-12 containing10% fetal bovine serum (PBS)) and down regulated with basic medium(DMEM/F-12 containing 2% PBS) 12 hours prior to use.

(ii) In Vitro Cytocompatibility Study

50 μl (50,000 cells) of the tenocyte cell suspension in basic medium wasadded to each collagen disc. After 1 h incubation at 37° C. and 5% CO₂atmosphere without medium emersion, the cell-seeded discs weretransferred to a 24-well plate prefilled with 2 ml of basic medium aloneor in combination with rhPDGF-BB (30 ng/ml). After 12 h static culture,the plates with cell-seeded discs were placed on an orbital shaker (60rpm) in the incubator. Medium changes were provided every 48 h. On days2, 4, and 6, triplicate samples from each material and treatment wereutilized for ATP assay, to determine the live cell number in/on thecollagen matrices. On day 4, one sample from each material and treatmentwas used for scanning electron microscopic (SEM) evaluation. On day 6,quadruplicate samples from each material and treatment were utilized forhistological assessment.

(iii) In Vivo Biocompatibility and Biodegradation Study

A total of 12 New Zealand white rabbits were used for this study.Through an anterior-medial incision, the femur was exposed. Afterremoving the periosteum, the midpoint of the ventral surface of thefemur was located and two areas (1 cm×1 cm), approximately 0.5-0.8 cmproximal and distal to the midpoint, were decorticated using a round burand copious irrigation to prevent overheating of the bone. Two holes(0.9 mm) were drilled at the midpoint of the decorticated site on themedial and lateral edges. Finally, a 5-0 silk suture was passed throughthe two holes and the collagen pad (pre-saturated with 100 μl of 0.3mg/ml rhPDGF-BB) was then tied to secure the pad to the surface of thefemur. Surgery was conducted bilaterally, so each femur received twopads and each animal received one each of the four different collagenpads. The order of pads on the femur was randomized. At 1, 2, and 3weeks post surgery, 4 animals were euthanized and the femurs, togetherwith the pads and surrounding tissue, were fixed in 10% neutral bufferedformalin (NBF), embedded in methyl methacrylate (MMA), and stained witha Goldner's trichrome procedure.

Results

(i) In Vitro Cytocompatibility Study

The ATP assay revealed that cell growth on/in the collagen discssignificantly increased in the presence of rhPDGF-BB, and increased withtime in culture, with no significant differences observed across thedifferent matrices under the same treatment conditions. The SEM imagesfor the matrices alone revealed qualitative differences in porosity withmatrix D demonstrating the greatest porosity and matrices A, B and Cshowing decreasing levels of porosity. SEM results for each of thematrices following cell seeding showed that there were more cellsobserved on the surface of the three fibrous matrices of differingdensities, with the fewest observed on the surface of the “highlyporous” matrix (D). Histological images from cross sections of each ofthe matrices seeded with cells further supported the SEM observations,demonstrating cell distribution along the outside surface of the padsfor matrices with increasing density, with matrix D demonstrating thegreatest distribution of cells throughout the matrix.

(ii) In Vivo Biocompatibility and Biodegradation Study

Histological assessment revealed that all four collagen pads remainedintact 1 week post-implantation. In the tissue around the four differentmatrices, there was increased cell infiltration of granulocytes andmononuclear cells. Two (2) weeks post implantation, partial degradationwas observed for matrices A, B, and D with obvious cell infiltrationassociated with matrix D. Three (3) weeks post implantation, matrix Dwas mostly degraded with only small collagen fragments observed amongnormal fibroblastic cells (FIG. 9D). Sites receiving collagen matrixderivatives of dermal origin (A, B, and C) exhibited degradation whichwas inversely proportional to the collagen density. Extensiveinflammatory cell infiltration was observed for these matrices whichbecame more localized over the three week observation period (FIGS.9A-9C).

The results showed that collagen matrix D was the most porous, with ahomogenous fine collagen fiber network, providing more accessiblesurface area for cell attachment and migration. In vivo evaluation ofthe individual matrices showed that collagen D exhibited the shortestresidence period and appeared to be the most biocompatible material,demonstrating minimal inflammatory cell infiltration 3 weekspost-implantation, while the collagen matrices of differing densitiesdemonstrated increasing inflammatory cell infiltration which localizedinto foci by 3 weeks post-implantation.

Example 9: Use of rhPDGF-BB and a Collagen Wrap to Augment TunnelFixation of Tendon in a Caprine Model of Tendon-Bone or Ligament-BoneReattachment

This study assesses the histological and biomechanical benefits ofwrapping a collagen sponge soaked in a dose of rhPDGF-BB prior toinsertion and fixation of the long flexor tendon in a transmetaphysealtibial tunnel. The method of affixing grafts for anterior curciateligament reconstruction is as described by Rodeo et al., J. Bone JointSurg. Am., 75(12):1795-1803 (1993). A collagen pad in combination withrhPDGF-BB is used to promote more rapid, complete integration of thetendon or ligament within a tibial insertion site.

(A) Materials and Methods

(i) Species

The goat is a suitable model because, like humans, its bones undergoremodeling, which is a balanced combination of bone formation andresorption leading to normal bone structure. The bones of smaller animalmodels, such as rats or mice, do not undergo remodeling and thus do notrepresent the biological processes that would occur as the tendonreintegrates with bone. Furthermore, the tendons of larger animals, suchas goat, can be more easily manipulated and reattached using techniquesand instrumentation used in human surgery.

A total of 24 skeletally mature goats (female, mixed genetic background)are used in this study. These animals are divided among 3 treatmentgroups (Table 7). Group 1 undergoes sharp detachment of the long flexortendon from its femoral insertion on the lateral side, followed bythreading through a bone tunnel drilled obliquely through the tibialmetaphysis. Once on the other side, the tendon is attached to the medialcortex of the tibia with stainless steel sutures (see FIGS. 10 and 11).The contralateral limb of Group 1 animals comprises those in Group 2.Group 2 undergoes the same surgical procedure, but prior to threadingthrough the tunnel, the tendon is wrapped with a 25×15 mm collagensponge (COLLATAPE®, Integra LifeScience Corporation, Plainsboro, N.J.).Once affixed to the medial cortex of the tibia, the collagen matrix ishydrated with 1.0 mL of sodium acetate buffer (20 mM, pH 6.0). Withineach animal, the limbs receiving these treatments are randomized (Table8). A total of 10 animals are used for Groups 1 and 2, resulting in ann=5 for each time point (2 and 4 weeks). Each time in life (the cohortof animals used for each time point and each treatment) produces 3specimens for biomechanical testing and 2 specimens for histology.

Group 3 animals undergoes the same surgical procedure as those in Group2 but the collagen sponge is hydrated with 1.0 mL of 1.0 mg/mLrecombinant human platelet-derived growth factor BB (PDGF-BB). In thisgroup, both limbs receive treatment with PDGF-BB. Group 3 consists of 10animals; this results in an n=10 samples (two operated limbs/animal) foreach time point (2 and 4 weeks). Each time in life produces 5 specimensfor biomechanical testing and 5 specimens for histology.

Two more animals are used for a pilot surgical study to confirmfeasibility of surgically implanting the material (one receivestreatment as described for groups 1 and 2 above and the other receivestreatment as described for group 3 above). These animals are observedfor 2 weeks post-surgery to confirm a return to normal ambulation and anabsence of complications. Following this period, animals from the pilotstudy are euthanized and the tissues collected. If the surgeries andpost-operative evaluative period occur without incident, the remainderof the study (20 animals) is performed as planned.

(ii) Test and Control Articles

Test Article 1 is 1.0 mg/ml rhPDGF-BB in 20 mM sodium acetate buffer, pH6.0+/−0.5, liquid form and stored at 2° C.-8° C. Test Article 2 iscollagen from COLLATAPE®, Integra LifeSciences, solid form and stored atroom temperature.

iii) Dose Preparation

Mixing of Collagen with rhDGF-BB

The test and control articles are mixed. Aseptic preparation is used forarticles. All mixing is performed at room temperature. The formulatedtest and control articles are used for up to 1 hour after preparation.

The collagen pads are cut to 25×15 mm, wrapped over the tendon, andthreaded through the bone tunnel. With a 27 G needle, 1.0 mL of 1.0mg/mL rhPDGF-BB is injected into the tunnel adjacent to the finalimplant site of the collagen matrix. The medial and lateral openings ofthe tunnel receive approximately 0.5 mL total of 1.0 mg/mL rhPDGF-BB.The injections made at either the medial or lateral sides of the tunnelare made by injecting at multiple points of the tunnel perimeter,roughly the ¼, ½, ¾, and full circumference of the tunnel opening.

(iv) Test System (Animal and Animal Care)

Skeletally mature female domestic goat (mixed breed, n=24), 30-40 lbs,Q-fever tested with health certificate, are used. No animals have beenused for prior experimentation. Animals are placed in runs with aminimum of 10 square feet of space per goat. Cages are constructed ofstainless steel and cleaned routinely. Ambient temperature is maintainedbetween 60-80° F. and humidity is maintained between 30-70%.

Goats are provided water ad libitum in a bucket as well as via anautomatic watering system (LIX-IT). Hay is provided continuously andonce per day goats are provided commercially purchased goat chow. Thestudy animals are acclimatized to their designated housing for at least14 days prior to the first day of dosing. This acclimation period allowsthe animals to become accustomed to the study room setting. All animalsare given a physical examination, including heart rate, respiration, andfecal floatation. Only those animals judged to be in excellent health bythe veterinarian are admitted to the facility and accepted for thestudy. Animals are treated prophylactically with Ivomec (1 cc/75 lbs)and Penicillin G and Benzocaine (1 cc/10 lbs).

(B) Experimental Design

(i) General Description and Surgical Method

Anesthesia is induced within 30 minutes of premedication using goatcocktail [10 cc ketamine (100 mg/ml)+lee xylazine (20 mg/ml)] at 1 ml/10kg IM. Following induction, a cephalic IV catheter is put in place.Ophthalmic ointment is gently placed on the cornea to minimize drying ofthe eye. Goats are intubated using an endotracheal tube (5-8 ETT) aswell as a rumentube. Anesthesia is maintained by use of 1.5-2%isoflurane delivered in 100% oxygen using a rebreathing circuit. Ifneeded, the goat is placed on a ventilator according to the tidal volume(15 mL/kg of BW). Each goat receives warmed Lactated Ringer's solutionat 10 ml/kg/hr throughout anesthesia.

Penicillin G and benzocaine (1 cc/10 lbs BW) is administered one hourprior to surgery and EOD (end of day) after surgery for a total of 3doses. Buprenorphine (Buprenex) is administered at 0.005 mg/kg BW IMBID×48 hrs post-op.

The surgical sites is prepared for aseptic surgery (3-chlorhexidine [4%]scrubs alternating with sterile saline solution) and draped.Aseptically, each knee joint is exposed by a lateral parapatellarincision, and the long digital extensor tendon sharply detached from itsinsertion on the lateral femoral condyle (FIG. 10). The proximal tibialmetaphysis is exposed by incision of the fascia of the anterior tibialismuscle and retraction of the muscle laterally.

A 5.6 mm diameter drill hole is made in the proximal tibial metaphysisat an angle between 30 and 45 degrees to the long axis of the tibia(FIG. 11). All drilling is performed with copious irrigation and beginsproximally at the medial tibial condyle (1 cm below the palpated jointline) and end distally on the lateral side of the tibia (2 cm below thepalpated joint line). The depth of the drilled hole is measured with athin metal depth gauge and recorded. A 25×15 mm collagen sponge iswrapped around the tendon without overlap, secured with 4-0 Vicrylsuture, and then threaded through the bone tunnel with stainless steelwire. Once on the other side of the bone, the tendon is fixed to themedial cortex by use of two small holes and interrupted sutures of 4-0stainless steel. After securing the collagen-wrapped tendon to the bone,1 mL of sodium acetate buffer pH 6.0 in divided doses is injected intoboth sides of the tunnel using a 27 G syringe needle to hydrate thecollagen sponge. The tendons of control animals, receiving no collagenwrap, is simply threaded through the bone tunnel and fixed on the medialcortex. The growth factor-treated tendons are wrapped with collagensponge as above, threaded through the bone tunnel, fixed on the medialcortex and then hydrated with 1 mL sodium acetate (pH 6) buffercontaining 1 mg/mL PDGF-BB.

Soft tissues are closed in layers with resorbable sutures (e.g. 4-0Vicryl). After closure of the incision and skin, a radiograph of eachlimb is performed to document tunnel location and angle. Ideally, theradiograph is oriented so as to image the entire length of the tibialtunnel

When goats are sternal and ambulatory, they are returned to their pens.Each goat is allowed to eat and drink ad libidum.

The addition of a dose of rhPDGF-BB to the portion of the long flexortendon inserted into the metaphyseal bone tunnel is expected to exhibit,over time, a 25% increase in biomechanical load to failure, and improvedintegration and mineralization by histology and micro-CT.

The surgical procedure is first performed on 2 animals in the presenceof the veterinarian to review all aspects of surgery and animal care.After 14 days, if all animals recover without incident, the experimentalprotocol is then implemented. In the event that difficulties arise, theinvestigators work diligently with the veterinary staff to resolve anyissues necessary to provide optimal care to the animals and ensure thesuccess of the study. During surgery, the animals will be anesthetizedand animals are sedated for blood sampling procedures.

(ii) Group Assignments and Dose Levels

TABLE 7 Treatment Groups Animals Growth Group (n) Operated Side MatrixFactor Sacrifice Endpoints 1 & 2 10 Randomized, Naked tendon or None 2weeks Histomorphometry (n = 2) Bilateral 25 mm × 15 mm 4 weeksBiomechanics (n = 3) collagen sponge 3 10 Bilateral 25 mm × 15 mm 1.0mg/mL 2 weeks Histomorphometry (n = 5) collagen sponge PDGF-BB 4 weeksBiomechanics (n = 5)

TABLE 8 Assignment to Treatments Left Right Animal # Group Limb Limb 1 1& 2 N SP 2 1 & 2 SP N 3 3 PDGF PDGF 4 3 PDGF PDGF 5 1 & 2 N SP 6 1 & 2SP N 7 3 PDGF PDGF 8 3 PDGF PDGF 9 1 & 2 N SP 10 3 PDGF PDGF 11 1 & 2 SPN 12 1 & 2 N SP 13 1 & 2 N SP 14 3 PDGF PDGF 15 1 & 2 N SP 16 3 PDGFPDGF 17 3 PDGF PDGF 18 3 PDGF PDGF 19 1 & 2 SP N 20 3 PDGF PDGF N: Nakedtendon SP: Tendon wrapped in collagen sponge (COLLATAPE ®) PDGF: Tendonwrapped in collagen sponge containing rhPDGF-BB(C) In-Life Observations and Measurements

Animals are observed at least daily throughout the study. Recording ofobservations commences once the pre-selection criteria are complete andcontinue until the end of study. Each animal is observed for changes ingeneral appearance and behavior, including changes in ambulation. Bodyweights is measured prior to surgery and at sacrifice. Body weights aretaken at additional timepoints as necessary to monitor the animals'health.

At the end of post-operative week 1, animals sacrificed at 2 weeks areinjected with 10 mg/kg calcein (vendor TBD) IP (intraperitoneal).Similarly, at 3 weeks, animals sacrificed at 4 weeks are injected with10 mg/kg calcein (vendor TBD) IP. These injections provide delineationof new bone formation and growth into the tunnel of histologicalspecimens.

(D) Clinical Pathology Evaluation

(i) Serum Collection for Analysis

Approximately 5 mls of blood are collected into non-additive (i.e.,“clot”) tubes from all animals once pre-surgically, and prior tosacrifice. The blood is centrifuged to obtain serum and divided into twoaliquots. The serum is stored at −70° C. or colder for further analysis.

(E) Anatomic Pathology

All animals are sacrificed at the appropriate study end points. A grossnecropsy is conducted on each animal found dead or sacrificed in amoribund condition to determine the cause of death.

(i) Necropsy

Goats are euthanized in accordance with the USDA Animal Welfare Act andThe Guide for Care and Use of Laboratory Animals (ILAR publication,1996, National Academy Press. Goats are first sedated using goatcocktail [10 cc ketamine (100 mg/ml)+lee xylazine (20 mg/ml)] at 1 ml/10kg IM. Then, Euthasol (360 mg/ml sodium pentobarbital) at 1 cc/10 lbs BWIV is given. Death is confirmed by auscultation and lack of reflexes(blinking, withdrawal, etc).

(ii) Tissue Collection and Preservation

Gross Observations and Photography of Implant Sites

At the time of euthanasia, the implant site is grossly examined and adescription of the site recorded. Digital photography is used todocument the observations.

(iii) Pathology

The stifle joint is disarticulated and a Stryker saw used to cut acrossthe tibia at the mid-shaft. The surrounding muscles and skin is trimmedas much as possible without exposing any more of the bone wound thannecessary to perform the clinical evaluation. Each specimen is labeledwith a goat number as well as an indication of whether it is the rightor left limb. Tissues are placed into neutral buffered formalin (10 volfixative: 1 vol tissue) and shipped to the sponsor.

(F) Endpoints

(i) Histology and Histopathology

Histological processing is conducted. In brief, the tissues are embeddedin methyl methacrylate (MMA), sectioned, and stained usinghematoxylin/eosin, Safranin O/Fast Green, Von Kossa/MacNeal's and/or VanGieson's for light microscopic evaluation. The plane of section forevaluation is as cross-section and longitudinal to the tibial tunnel. Agrading system is devised to assess the amount of material remaining ateach implant site, extent of mineralization of the tendon and amount ofnew bone growth along the tunnel walls. Calcein visualization isperformed on unstained sections adjacent to sections stained withhematoxylin/eosin

(ii) Biomechanics

Specimens retained for biomechanical testing is frozen to −20° C. untiluse. Tibia/tendon complexes are secured to a multiaxial table in orderto align the long axis of the tunnel with the direction of the pulledtendon. This orientation minimizes any effect of friction and allows fordirectly testing tendon integration/mineralization with the surroundingbone. Peak load to failure measurements is normalized to the length ofthe tunnel measured by hand and post-operative radiographs.

(a) Study Design

The study determines the mechanical performance of a simulated digitalextensor tendon reattachment in a tibial bone tunnel. 12 Boer goats areutilized for this study.

(b) Biomechanical Testing

Specimens are wrapped in saline soaked gauze and stored at −20° C. untiltesting. The distal portion of the tibia is potted in 2″ PVC pipe usinghigh polymethylmethacrylate. Specimens are kept moist during the pottingpreparation and biomechanical testing with a saline spray at 15 minuteintervals. The potted tibae are mounted in a custom-designed testingfixture that is rigidly attached to the materials testing system loadingframe (MTS MiniBionix, Edan Prairie, Minn.; FIG. 12). A custom-designedcryo-clamp designed to grab the natural cross section of the tendon isused in this study to apply uniaxial traction forces to the construct.Any remaining lateral sutures are transected.

Analysis of the biomechanical and histological readings is carried outthrough analysis of covariance techniques. The effects of rhPDGF-BB andCOLLATAPE® are investigated using animal body mass and tendon diameteras covariates.

Readings may be transformed prior to analysis through appropriatemethods.

Phase 1: 30 Cycle Dynamic Preconditioning

A cyclic loading test is initially employed to precondition the tendonrepair. A 10 Newton (N) preload is applied and the construct force isallowed to relax by approximately 40%. This is designated the initialconfiguration for all constructs. The repaired construct is thencyclically preconditioned in a force-control protocol from 10 to 30 N at0.25 Hz for 30 cycles to reach a steady-state. Thirty (n=30) cycles ischosen because previous experiments in our laboratory demonstrate thatthe slope of the displacement versus time curve appears to become stablebetween 20 and 30 cycles.

Phase 2: Quasi-Static Failure Loading

Following preconditioning, the repaired constructs are loaded to failureunder displacement control at a rate of 1 mm/s. Biomechanical parametersof interest include ultimate load-to-failure and quasi-static stiffness(defined as the slope of the load-displacement curve). Finally, thefailure mechanism is documented for each specimen. Digital images aretaken of each specimen as loaded into the testing device and followingfailure to document mode and condition of the failure.

(iii) Microtomography (CT)

Each specimen is scanned at an optimal resolution determined by specimensize. See FIG. 13. Slices are acquired at approximate cross section tocylindrical defect. A semi-automated contouring method is used to selecta volume of interest (VOi) limited to the perimeter of the originalborders of the defect. An optimized density threshold and noise filterare selected and applied uniformly to all specimens to segment bone fromsoft tissue. The total mean density, mean density of bone, and bonevolume/total volume within tunnel are calculated.

Example 10: Evaluation of the Release Characteristics of rhPDGF-BB(Recombinant Human Platelet-Derived Growth Factor-BB) from BIOBLANKET™and COLLATAPE® Matrices by BenchTop Model

The objective of this study is to measure rhPDGF-BB release fromBIOBLANKET™ matrices having different densities and COLLATAPE® matrixusing a BenchTop model at room temperature.

Test Materials

TABLE 9 BIOBLANKET ™ and COLLATAPE ® Matrices Sample Lot # BIOBLANKET ™Collagen (5%) R436-1 BIOBLANKET ™ Collagen (6%) R436-2 BIOBLANKET ™Collagen (7%) R436-3 COLLATAPE ® Collagen 1080464Study Design

The study design is listed in Table 10, showing initial flushing time of5 minutes for all the sample groups (BIOBLANKET™ matrices, COLLATAPE®matrix, and control group without the collagen matrix.

TABLE 10 Study Design Sample Test Materials Initial Flushing Time 1-3BIOBLANKET ™ Collagen (5%) 5 min 4-6 BIOBLANKET ™ Collagen (6%) 5 min7-9 BIOBLANKET ™ Collagen (7%) 5 min 10-12 COLLATAPE ® 5 min 13-15Control (rhPDGF-BB only) 5 min

Using aseptic technique, 8-mm disks are punched from each of theBIOBLANKET™ and COLLATAPE® sheets with a biopsy punch. A syringe needleis used to gently impale one BIOBLANKET™ and COLLATAPE® disk on a 27 G1¼needle and connect the needle with 1 mL syringe head which is installedin a specially designed chamber. 50 μl of PDGF-BB (1.0 mg/mL in 20 mMsodium acetate buffer) is applied to each disk. The disks are thenincubated at room temperature for 10 minutes. One end of the silicontubing to the arthroscopic cannular device and the other end of thetubing to 20 ml syringe are connected as shown in FIG. 14. 20 ml elutionbuffer (EME+2% PBS) is filled into the syringe, and the silicon tubingto the Varistaltic pump is assembled. The collagen pad saturated withrhPDGF-BB is loaded to the top the arthroscopic cannular device. Theflow rate at 200 ml/min (precalibrated) is set up. Pump turns on andruns for 5 minutes. For the control, 50 μl of rhPDGF-BB is added at 1.0mg/ml to the system from the top of arthroscopic cannular device. After5 minutes of flushing, collect the elution buffer in a 50 ml conicaltube by disconnect the silicon tubing from the 20 ml syringe while thepump still runs. The samples are stored at 2-4° C. for analysis. Theamount of rhPDGF-BB eluted in each sample is measured using the DuoSetELISA kit from R & D systems, as described in ELISA Assay Procedurebelow.

ELISA Assay Procedure

The capture reagent is diluted to the working concentration (0.4 μg/ml)in DPBS by adding 56 μl of capture reagent stock solution to 10 ml DPBS,100 μl of diluted capture reagent is then added to each well of a96-well plate. The plate is sealed with plate sealer, and incubated atroom temperature overnight on a shaker. The bubbles from the Aspirationand dispensing manifold are drained and plates are washed three timeswith wash buffer. 200 μl of Elution Buffer are added to each well andplates are blocked for at 2 hours (maximum 4 hours) at room temperaturewith rocking. Plates are washed three times with wash buffer.

Steptavidin-HRP is diluted to the working concentration (200× dilution)in Reagent Diluent Buffer by adding 50 μl of Streptavidin-HRP stocksolution to 10 ml Reagent Diluent Buffer. The Steptavidin-HRP is thenadded in 100 μl to each well and covered with aluminum foil, andincubated for 20 minutes at room temperature on orbital shaker.Immediately after adding Streptavidin-HRP to plate, needed volume ofSureBlue TMB is then aliquoted into a 15 ml conical tube wrapped in foiland placed on bench to allow equilibration to room temperature. Separatetube for each plate is prepared. Plates are then washed three times withwash buffer.

Sure Blue is added in 100 μl to each well, covered with aluminum foil,and incubated 20 minutes at room temperature. 50 μl IN HCL are thenadded to each well to stop the reaction. The optical density at 450 nmare read with a correction set at 540 nm within 30 minutes after thereaction is topped.

Data Calculation

Standards using the 4-Parameter graph are plotted, and rhPDGF-BBconcentration for each test sample at each dilution using the standardcurve on each plate is calculated. Mean values and standard deviations(SD) for each of the triplicate samples from the two dilutions at eachtime point is also calculated.

The total rhPDGF-BB present in each sample is determined by multiplyingrhPDGF-BB concentration with the total volume of each sample. Thecumulative amount of rhPDGF-BB in each sample at each time point iscalculated by summing the amount of rhPDGF-BB at that time point to theprevious time point.

Mean+/−SD for cumulative amount of rhPDGF-BB released at each of thefour time points is plotted. The percentage of rhPDGF-BB release in eachsample is calculated by dividing the cumulative rhPDGF-BB release ateach time point with the mean value of control at the same time point.The mean values of percentage of rhPDGF-BB release for each of thetriplicate samples at each time point are also calculated. The mean+/−SDfor percentage of rhPDGF-BB release at each of the four time points isplotted.

Statistical Analysis

Statistical comparisons of data at each time point are done by anappropriate method according to the data distribution.

Example 11: Characterization of Release, Stability, and Biopotency ofRecombinant Human Platelet-Derived Growth Factor-BB (rhPDGF-BB) Combinedwith Collagen Matrices Using a BenchTop Model of Arthroscopic Irrigation

This study was conducted to develop a novel Bench Top Arthroscopic Modelto replicate the high fluid flow arthroscopic environment, and tocharacterize the release, stability, and biopotency of rhPDGF-BB, elutedfrom four different collagen matrices considered for application intendon to bone attachment procedures, such as anterior cruciate ligamentreconstruction procedure or rotator cuff injuries treatment procedure.

Methods

Four Type I bovine collagen matrices were evaluated: three dermallyderived, collagen matrices of differing collagen concentrations (A=4.5%collagen; B=5% collagen, C=6% collagen from Kensey Nash Corporation) andone Achilles tendon derived matrix (collagen D, COLLATAPE® from IntegraLifeSciences). All matrices were punched into 8 mm discs. To assess therelease of rhPDGF-BB (Novartis), from the collagen matrices, each discwas hydrated with 50 μl of rhPDGF-BB at 1 mg/ml (50 μg), incubated for10 min at room temperature, loaded into the Bench Top Arthroscopicsystem (see FIG. 14) prefilled with 20 ml of elution buffer (MEMcontaining 2% PBS), and flushed for 5 min at 200 ml/min flow rate. Thesame amount of rhPDGF-BB was added to the system as a control. Elutionbuffer samples used to wash the matrices were analyzed using a DuoSetELISA assay (R & D Systems). Reversed phase and size exclusion highperformance liquid chromatography (HPLC) was used to derive profiles ofrhPDGF-BB released from the collagen matrices for assessment ofchanges/modification in the native/denatured structure of rhPDGF-BB. Thebiopotency of rhPDGF-BB released from the collagen matrices was testedusing a bromodeoxyuridine (BrdU) cell proliferation assay (Promega).NIH3T3 fibroblasts were cultured in releasate containing rhPDGF-BB(0-0.24 μg/ml), BrdU was then added, and the cells incubated for 48hours.

The mean percent rhPDGF-BB release, relative to control for each of thefour collagen matrices was as follows: collagen A, 79%; collagen B,64.6%; collagen C, 74.3%; and collagen D, 89.0%. Collagen D showed thegreatest release of rhPDGF-BB, which was significantly greater than thatobserved for matrix B or C as demonstrated using a One Way Analysis ofVariance Fisher LSD Method. No apparent changes to the rhPDGF-BB werefound following combination with matrix D, as demonstrated by reversedphase HPLC, but mild rhPDGF-BB oxidation occurred with collagen matricesA, B, and C, however these changes did not appear to affect biopotency(as evaluated by BrdU).

This study shows that the Bench Top Arthroscopic model is an effectivesystem to evaluate matrices for the delivery of recombinant proteintherapeutics proposed for use in sports medicine arthroscopicrepair/regenerative procedures. Collagens A and D released morerhPDGF-BB than did collagens Band C, and collagen D did not result inany changes to the rhPDGF-BB, exhibiting great potential for use insports medicine regenerative procedures. Further, this arthroscopicmodel represents an excellent in vitro tool which allows one to tailorrecombinant protein therapeutic devices to provide the optimal deliverydose for maximum effectiveness.

Example 12: Cell Migration in BIOBLANKET™ Matrices of DifferentDensities and COLLATAPE® in Response to rhPDGF-BB Via a ScanningElectron Microscopic (SEM) Assessment

This study assessed the extent of cell migration into BIOBLANKET™ andCOLLATAPE® matrices by culturing primary ovine tenocytes that aretreated with or without rhPDGF-BB and subsequently assessed via scanningelectron microscopic (SEM) technique.

Materials and Methods

The migration of ovine tenocytes into the BIOBLANKET™ matrices andCOLLATAPE® was evaluated by seeding the matrices with a known quantityof cells and then culturing the matrices for 4 days. The matrices werethen processed by critical point drying and the cell distribution anddensity in the matrices were assessed under scanning electronmicroscope.

The test materials include 1) 5%, 6%, or 7% BIOBLANKET™ (Lot# R436-1,R436-2, R436-3); 2) COLLATAPE® (Lot#1072549); 3) rhPDGF-BB (0.3 mg/ml;Lot#: BMTI204), and 4) fresh ovine foot, amputated at the ankle joint.The study design is listed in Table 11.

TABLE 11 Sample Layout Cell suspension Cell suspension (P+)″ (P−)″Sample Medium Medium Medium Medium No. Materials (P+)′ (P−)d (P+) (P−)01 5% Collagen 02 5% Collagen 03 5% Collagen 04 5% Collagen 05 6%Collagen 06 6% Collagen 07 6% Collagen 08 6% Collagen 09 7% Collagen 107% Collagen 11 7% Collagen 12 7% Collagen 13 COLLATAPE ® 14 COLLATAPE ®15 COLLATAPE ® 16 COLLATAPE ® aSuspension (P+): the cell seedingsuspension contains 1.2 mg/ml rhPDGF-BB bSuspension (P−): the cellseeding suspension does not contain rhPDGF-BB ^(o)Medium (P+): cellculture medium contains 30 ng/ml of rhPDGF-BB dMedium (P−): cell culturemedium does not contain rhPDGF-BB(i) Isolation and Culture of Primary Ovine Tenocytes

Fresh ovine foot was cleaned, sprayed and washed using soap, water, and70% alcohol. About 3 inch wide of skin from the surface of flexor tendonwas excised. The incision was sprayed with 70% alcohol and covered withsurgical drapes. An incision over the tendon sheath was made to exposethe tendon. The tendon was then severed from distal side. The tendon waspulled as long as possible, and the proximal side was severed. Tendonwas then placed in a 50 ml conical tube filled with ice-cold DPBS. Thefoot and waste tissue were discarded.

The tendon tissue was minced to small pieces in a sterile 120 mm cellculture dish in a laminar flow hood working area. The minced tissue wasthen transferred to a fresh 50 ml conical tube. The minced tendon tissuewas washed twice with DPBS and once with DMEM/F-12 medium. Tendon tissuewas then digested with 500 Units/ml Pronase protease in DMEM/F-12 mediumfor hour. The pronase protease was aspirated and washed twice with DPBSand once with serum-free DMEM/F-12 medium.

The tenocytes were then liberated with 0.2 collagenase P plus. 150Units/ml DNAse-11 in serum free DMEM/F-12 medium for one hour. Thedigest was filtered through a 75-μm cell strainer. The tissue left wasreturned in the strainer to a 50 ml conical tube, and the tenocytes wereliberated with 0.2% collagenase P plus 150 Units/ml DNAse-II in serumfree DMEM/F-12 medium for one hour. The cells were pelleted bycentrifugation at 1200-1500 RPM for 5 minutes at 4° C. The cells wereresuspended in 10 ml of DMEM/F12 growth medium and a cell count wasperformed using Trypan blue and hemacytometer. The cells were plated inT75 or T150 flasks with DMEM/F-12 growth medium at a density between5000-7500 cells/cm². The entire process described in this paragraph wasrepeated every one hour until all the tendon tissues were digested. Thegrowth medium was changed every two days and changed to basic medium 24hours prior to seeding the cells to the BIOBLANKET™ and COLLATAPE®matrices.

(ii) Cell Seeding and rhPDGF-BB Addition

Using sterile technique, 8 mm discs from the BIOBLANKET™ and COLLATAPE®matrices were punched using a biopsy punch. One BIOBLANKET™ orCOLLATAPE® disc on each 27 G ½ needle was gently impaled and the needlewas bent 90 degree angle twice to an open rectangular shape to securethe disc form sliding down. The needle impaled with BIOBLANKET™ andCOLLATAPE® matrices disc were connected to 1 ml syringe head in thespecially designed chamber. Tenocytes were trypsinized and suspended(less than 4 passages) in 1 ml of DMEM basic medium containing 2% PBSand antibiotics at a concentration of 10⁶ cells/ml. A total amount of 60ng (30 ng/ml in 2 ml medium) of rhPDGF-BB was loaded to each disc.BIOBLANKET™ and COLLATAPE® matrices discs were then seeded with cellsand incubated in the chamber in the incubator without media immersionfor 1 hour. DMEM medium containing 2% PBS was prepared and added in 2 mlto 8 wells of each 24-well ultra low attachment plate for total 8 wellsas rhPDGF-BB treated group. After 1 hour incubation, the cell-seededdiscs from the loading boxes were removed. By using hemostat, the needletip from the plastic root was broken. The cell-seeded discs weretransferred together with the needle tip to wells prefilled with mediacontaining different compositions. The needle tip attached in the discskept the disc floating in the medium, so the cells on both sides of thedisc were fed evenly with nutrition. Total four treatments wereconducted for each material and triplicate samples were prepared foreach material and each treatment. After 12 hours' static culture in theincubator at 37° C. and 5% CO₂ atmosphere, the plates were placed withcell-seeded collagen matrices on an orbital shaker in the incubator. Themedium containing the same compositions as the first time feeding waschanged every 48 hours.

(iii) Scanning Electron Microscopic Process

After four days of culture, each cell-seeded disc from 24-well plate wastransferred to a cryovial with emersion of medium. The medium from thecryovial was then removed and the samples were washed in DPBS twice. Thesamples were fixed in 2.5% glutaraldehyde for 2 hours. The samples werethen washed in DPBS five times and post-fixed in 2% osmium tetroxide fortwo hours. The samples were soaked in deionized water for 10 minutes andwashed five times with deionized water to remove excess osmiumtetroxide. The samples were dehydrated in an ascending series of ethanoland then dried in a Polaron critical point drier. The samples werecoated with gold-palladium and viewed with a Hitachi SEM.

Results and Conclusions

The tenocytes were grown on the surface of all BIOBLANKET™ matrices withdifferent concentration of collagen slurry, but grown inside theCOLLATAPE® matrix. Most of the tenocytes were in round shape whilegrowing on all BIOBLANKET™ matrices with different concentration ofcollagen slurry but in spindle shape on the COLLATAPE® matrix. See FIG.15.

Example 12: Rotator Cuff Repair Using rhPDGF-BB and Type I BovineCollagen Matrix in an Ovine Model

The purpose of this study was to determine the efficacy of an rhPDGF-BBladen matrix intended to promote stronger reattachment of theinfraspinatus tendon to the humerus for rotator cuff repair using anovine model. The experimental design is provided as follows: 1) Sutureonly (n=9); 2) Suture+Collagen Matrix+Buffer (n=9); 3) Suture+CollagenMatrix+0.15 mg/ml (or 75 g) rhPDGF-BB (n=9); 4) Suture+CollagenMatrix+0.30 mg/ml (or 150 g) rhPDGF-BB (n=9); 5) Suture+CollagenMatrix+1.0 mg/ml (or 500 g) rhPDGF-BB (n=9); and 6) iCTL (n=9) IntactContra Lateral Control. 9 Animals from suture only, suture+CollagenMatrix+buffer, and the three dose groups were utilized for biomechanicaltesting, and 3 each for histology testing. The iCTL group was smallerwith 6 animals for biomechanical testing and 3 for histology testing.

Surgical Procedure

The infraspinatus tendon of skeletally mature sheep (3.5+ years) wassurgically exposed and sharply detached from the humeral head. Thetendon footprint was decorticated and three perforations were made intothe bone to induce bleeding. The test articles were placed as aninterpositional graft between the tendon and the bone. Two sutures werepassed through the tendon using a Mason-Allen technique and the tendonwas secured to the humeral head through a single-row repair consistingof 3 bone tunnels. The surgical site was closed using standard procedureand the sheep were allowed to ambulate normally. Animals were sacrificed12 weeks after surgery.

Materials and Methods

Biomechanical Testing

Shoulders from animals allocated for biomechanical testing wereharvested and denuded of all musculature, while leaving thehumerus-infraspinatus tendon construct intact. A total of 51 shoulderswere biomechanically evaluated. Following cleaning, specimens werewrapped in saline soaked gauze and stored at −20° C. until biomechanicaltesting. The humeri were potted in 2″ PVC pipe using high strengthpoly-methyl-methacrylate (PMMA) resin. Specimens were kept hydratedduring the potting preparation and biomechanical testing with a salinespray at 15 minute intervals. The potted humeri were mounted in acustom-designed testing fixture that was rigidly attached to thematerials testing system loading frame (MTS MiniBionix, Edan Prairie,Minn.). A custom-designed cryoclamp which was designed to preserve thenatural cross section of the infraspinatus tendon and minimize softtissue slippage was to apply a uniaxial traction forces to the constructat an angle of approximately 135° to the potted humerus. This was doneto mimic the physiological force vector of the tendon. Testing commencedwhen a thermocouple attached to the cryoclamp registered −22° C., atemperature previously reported to be sufficient to ensure securecoupling between the tendon and clamp.

Three retroreflective markers were sutured or glued onto the pottedconstruct: one on the humerus immediately adjacent to the repair site,one on the tendon proximal to the repair interface, and the third on thecryoclamp. Three cameras (Motion Analysis, Santa Rosa, Calif.) recordedthe spatial movement of the markers at 60 Hz. Marker displacementmeasurements using the camera system allowed for real-time monitoring oflocal tissue deformation across the rotator cuff repair site.

Phase 1: 30 Cycle Dynamic Preconditioning

A cyclic loading test was initially employed to precondition the rotatorcuff repair. A 10 Newton (N) preload was applied in force control fortwo minutes, following which the repaired construct were cyclicallypreconditioned in a force-control protocol from 10 to 50 Nat 0.25 Hz for60 cycles to reach a steady-state condition. Sixty (n=60) cycles waschosen based on pilot experiments in our laboratory that havedemonstrated that the slope of the displacement versus time curvereaches a repeatable steady-state behavior between 50 and 60 cycles.Conditioning elongation and peak-to-peak elongation were determinedduring the cyclic preconditioning test. Conditioning elongation wasdefined as the distance in y-displacement between the 1st cyclic peakand the 60th cyclic peak. Peak-to-peak elongation was defined as theaverage of the local minimum to maximum of the 58th, 59th, and 60thcycles.

Phase 2: Quasi-Static Failure Loading

Following preconditioning, the repaired constructs were loaded tofailure under displacement control at a rate of 1 mm/s. Biomechanicalparameters of interest included ultimate load-to-failure andquasi-static stiffness (defined as the slope of the load-displacementcurve). Finally, the failure mechanism was documented for each specimen.Digital images were taken as appropriate.

Statistical Analysis

A One-Way ANOVA and post-hoc Fisher's LSD and Tukey test were used toidentify significant differences in continuous biomechanical parametersbetween treatment groups excluding the intact controls. Significance wasset at p<0.05 and all analyses were performed with SigmaStat 3.1 (SystatSoftware, Inc., San Jose, Calif.).

Results

Raw data from the cyclic preconditioning component of testing arepresented in Table 12. The 0.15 mg/ml PDGF and 0.30 mg/ml rhPDGF-BBgroups underwent significantly greater conditioning elongation than theSuture Only and Suture+Collagen Matrix groups (Tukey's: p≤0.024; FisherLSD: p≤0.003). FIG. 16A. There were no significant differences inpeak-to-peak elongation between any groups (p=0.111, FIG. 16B).

TABLE 12 Summary Data from Cyclic Preconditioning Analysis. DataReported as Mean ± S.E.M. Conditioning Peak-to-Peak ElongationElongation Treatment n (mm) (mm) Suture Only 9 0.510 ± 0.058 0.765 ±0.056 Suture + Collagen Matrix + 9 0.492 ± 0.086 0.729 ± 0.055 BufferSuture + Collagen Matrix + 9 1.069 ± 0.103 0.908 ± 0.051 0.15 mg/ml PDGFSuture + Collagen Matrix + 9 0.971 ± 0.119 0.841 ± 0.028 0.30 mg/ml PDGFSuture + Collagen Matrix + 9 0.689 ± 0.133 0.913 ± 0.086 1.0 mg/ml PDGFiCTL 6 0.070 ± 0.017 0.354 ± 0.030

Raw data from the ramp to failure component of the biomechanical testingare presented in Table 13. Repair augmentation with 0.15 mg/ml and 0.30mg/ml rhPDGF-BB resulted in a 63.7% and 63.3% increase in load tofailure relative to the Suture Only group, respectively (Tukey: p=0.176;Fischer LSD: p=0.029 and Tukey: p=0.181; Fisher LSD: p=0.030, FIG. 17A).Further, load at failure data indicated that the lower rhPDGF-BB dosesof 0.15 mg/ml and 0.30 mg/ml outperformed the higher 1.0 mg/ml PDGFdose, manifested as a 120% and 119.3% increase, respectively, in load atfailure (p=0.023 and p=0.023 (Fisher: p=0.003)). No statisticaldifferences in construct stiffness were identified between groups(p=0.254, FIG. 17B). Constructs in the 0.15 mg/ml and 0.30 mg/mlrhPDGF-BB group exhibited significantly greater elongation at failurerelative to the Suture Only group (p≤0.018 and p<0.024, respectively).Fisher's LSD indicated that the 0.15 mg/ml and 0.30 mg/ml PDGF groupselongated significantly more that the Suture+Collagen Matrix Buffergroup (p=0.015 and p=0.011, respectively). No differences in elongationat failure were identified between the 0.15 mg/ml, 0.30 mg/ml or 1.0mg/ml rhPDGF-BB groups (p≥0.054).

TABLE 13 Summary Data from Ramp to Failure Testing. Data Reported asMean ± S.E.M. Global Peak Peak Force Stiffness Elongation Treatment n(N) (N/mm) (mm) Suture Only 9  910.39 ± 156.13 131.41 ± 14.23  8.96 ±0.62 Suture + Collagen 9 1120.36 ± 157.43 157.33 ± 18.99 10.16 ± 1.01Matrix + Buffer Suture + Collagen 9 1490.51 ± 224.51 134.44 ± 15.5315.17 ± 0.98 Matrix + 0.15 mg/ml PDGF Suture + Collagen 9 1486.59 ±228.95 147.47 ± 13.71 15.38 ± 2.35 Matrix + 0.30 mg/ml PDGF Suture +Collagen 9  677.75 ± 105.94 104.25 ± 21.36 11.48 ± 1.34 Matrix + 1.0mg/ml PDGF iCTL 6 4211.61 ± 229.57 300.31 ± 17.68 16.79 ± 1.30

For the Suture Only, Suture+Collagen Matrix+Buffer, and Suture+CollagenMatrix+1.0 mg/ml rhPDGF-BB treatment groups, construct failure in everyspecimen was manifested as mid-substance tissue failure at the insertionsite on the humerus. No humeral avulsion failures were noted in any(n=27) of the tendons in these three groups. In contrast, failure modesin the 0.15 mg/ml rhPDGF-BB and 0.30 mg/ml rhPDGF-BB treatment groupswere mixed, manifesting as either mid-substance tissue failure at theinsertion site on the humerus or mid-substance tissue failure combinedwith some bony avulsion. Specifically, 6 of 9 (66.7%) of the shouldersin the 0.15 mg/ml rhPDGF-BB group exhibited some degree of bony avulsionwhile 5 of 9 (55.6%) of the shoulders in the 0.30 mg/ml rhPDGF-BB groupexhibited some degree of bony avulsion. Failure of the intact,contralateral constructs was manifested as either middiaphyseal humoralfracture (n=5) or bony avulsion at the infraspinatus tendon insertionsite on the humerus (n=1).

Conclusion

Augmentation of a humoral infraspinatus tendon reattachment with a 0.15mg/ml and 0.30 mg/ml of rhPDGF improved mechanical function after threemonths in an ovine model relative to the Suture Only, Suture+CollagenMatrix+Buffer and Suture+Collagen Matrix+1.0 mg/ml rhPDGF-BB groups.Enhanced biomechanical integrity of the lower doses of rhPDGF-BB wasmanifested as a 63% increase in load to failure relative to the SutureOnly group and 120% increase in load at failure relative to the 1.0mg/ml rhPDGF-BB group. The data reported here support a dose-dependenteffect on rotator cuff augmentation, with lower rhPDGF-BB doseseliciting a greater healing response relative to the higher 1.0 mg/mldose of the growth factor. Further, failure modes in the 0.15 mg/mlrhPDGF-BB and 0.30 mg/ml rhPDGF-BB treatment groups were similar to thefailure mode consistently seen in the intact, non-operated shoulders, as6 of 9 (66.7%) of the shoulders in the 0.15 mg/ml rhPDGF-BB groupexhibited some degree of bony avulsion while 5 of 9 (55.6%) of theshoulders in the 0.30 mg/ml rhPDGF-BB group exhibited some degree ofbony avulsion. This finding show that the lower doses of PDGF (e.g.,rhPDGF-BB) promoted greater tendinacious integration with the humoraltuberosity over the course of the 12 week healing period and that thelower doses of PDGF are more suitable for augmenting rotator cuffrepairs.

Example 13: Rotator Cuff Repair Using rhPDGF-BB and a Type I BovineCollagen Matrix in an Ovine Model: Histological Results

This study was designed to assess the effectiveness of Recombinant HumanPlatelet-Derived Growth Factor-BB (rhPDGF-BB) in combination with a TypeI Bovine Collagen matrix to promote healing and regeneration of thesheep rotator cuff insertion. Optimal healing of rotator cuff injuriesinvolves reinsertion of the tendon into bone at the original site ofattachment (tendon “footprint”). Without reinsertion of the tendonfibers into bone, the healed site is considered “weaker” than theoriginal attachment, potentially limiting function and leading togreater chances for re-injury. Previous studies have reported arelatively high rate of failure following rotator cuff repair (See,e.g., Boileau P., et al., J Bone Joint Surg Am, 87:1229-1240 (2005);Galatz L. M., et al., J. Bone Joint Surg Am., 86:219-224 (2004));Gazielly D. F., Clin. Orthop Relat Res, 304:43-53 (1994)); Gerber C. J.et al., Bone Joint Surg Am, 82:505-515 (2000); and Harryman D. T., etal., J. Bone Joint Surg Am, 73:982-989 (1991)), which has beenpostulated to result from a variety of different factors (see, e.g.,Goutalier D., et al., Clin Orthop, 304:78-83 (1994); Gerber C. et al., JBone Joint Surg Br, 76:371-380 (1994); Warner J. P., et al., J BoneJoint Surg Am, 74:36-45 (1992)). Tendon tissue quality andtendon-to-bone healing have been proposed as two of the most importantfactors contributing to failed rotator cuff repairs, and the delivery ofgrowth factors or cells to augment tendon-to-bone healing have beensuggested as methods to optimize healing of these injuries (see, e.g.,Gamradt S. C., et al., Tech in Orthop, 22:26-33 (2007) and Dovacevic D.,et al., Clin. Orthop Relat Res, 466:622-633 (2008)). PDGF-BB is a wellcharacterized wound healing protein which is known to be chemotactic(cell migration) and mitogenic (cell proliferation) for cells ofmesenchymal origin, including bone (osteoblast) and tendon (tenocyte)cells. Additionally, PDGF-BB has been shown to upregulate vascularendothelial growth factor (VEGF), leading to increased angiogenesis(revascularization), which is essential for successful regenerativeprocesses. The purpose of this study was to determine efficacy ofrhPDGF-BB, combined with a Type I Bovine Collagen matrix, at the site oftendon repair to augment and improve tendon reattachment usingbiomechanical and histological outcome measures.

Study Design

A total of 17 skeletally mature ovine were included as part of thestudy. Animals underwent surgical detachment followed by immediatereattachment of the right infraspinatus tendon, to the humeral greatertuberosity, using sutures through bone tunnels. In the first set ofexperiment, the experimental animals (n=3) received a type I collagencarrier combined with rhPDGF-BB with concentrations of 0.15 (n=3) or 0.3(n=3) mg/ml in 20 mM sodium acetate (Acetate) buffer at the tendon-boneinterface. Survival time for all animals was 12 weeks. Treatmentallocations are presented in Table 14.

TABLE 14 Specimens allocated for histology Survival Animals TimerhPDGF-BB Treatment Group (n) (weeks) Dose Endpoint Suture + Collagen 312 0.15 mg/ml Histology Matrix + 0.15 mg/ml rhPDGF-BB Suture + Collagen3 12 0.3 mg/mL Histology Matrix + 0.3 mg/ml rhPDGF-BB

In the second set of experiment, the experimental animals (n=3/group)received a type I collagen carrier (collagen matrix) combined withrhPDGF-BB with concentration of 1.0 mg/mL or collagen matrix alone in 20mM sodium acetate (Acetate) buffer at the tendon-bone interface. Table15.

TABLE 15 Specimens Allocated for Histological Evaluation TreatmentSample Size Suture Only n = 3 Suture + Collagen Matrix + n = 3 AcetateBuffer Suture + Collagen Matrix + n = 3 rhPDGF-BB (1.0 mg/mL) IntactControl n = 2 Total Number of Specimens  n = 11 for HistologyTissue Harvest and Trimming

Animals were humanely euthanized after 12 weeks of healing and operated(right) shoulders were harvested and placed in 10% neutral bufferedformalin for histological processing. Each shoulder was bisected throughthe infraspinatus tendon and its humeral attachment site into cranialand caudal halves using a scalpel for the tendon and a diamond blade sawfor the humerus (Exakt Technologies, Oklahoma City, Okla.). Digitalimages were taken of each specimen during trimming. One half (either thecranial or caudal aspect) was processed for decalcified histology; theother half was processed for undecalcified histological analysis.

Decalcified Histological Processing

Either the cranial or caudal aspect was processed for decalcifiedhistology and embedded in paraffin. The specimens were fixed,decalcified, dehydrated, cleared, infiltrated, and embedded usingstandard paraffin histology techniques and equipment (Sakura Tissue TEKV.I.P. Processor, Sakura Finetek USA, Inc., Torrance, Calif. and ShandonHistocentre 2, Thermo Shandon, Inc, Pittsburgh, Pa.). The paraffinblocks were faced and approximately 8 μm sections cut on a ShandonFinesse rotary microtome (Thermo Shandon, Inc, Pittsburgh, Pa.). Fivehistological sections were obtained from each shoulder at spatialthickness increments of approximately 250 microns. High-resolutiondigital images were acquired field by field for the entire stained slideand regions of interest using an Image Pro Imaging system (MediaCybernetics, Silver Spring, Md.), a Nikon E800 microscope (AG Heinze,Lake Forest, Calif.), and a Spot digital camera (Diagnostic Instruments,Sterling Heights, Mich.), and a Pentium IBM-based computer with expandedmemory capabilities (Dell Computer Corp., Round Rock, Tex.).

Semi-Quantitative Histopathology

All tissue sections were graded according to a grading scale to assessthe degree of tendon retraction (if any), evaluation of thereparative/healing tissue, the tendon bone interface, tissue response tothe treatments, vascularization, inflammation, collagenorientation/fiber alignment, and interdigitation and presence ofSharpey's fibers at the insertion site. Sections were first assessedblinded to treatment and evaluated for overall healing compared to oneanother and given a healing score. A description of criteria for eachscore is presented in Table 16. The degree of tendon retraction (if any)was also measured via calibrated gross digital images using Image ProPlus imaging system (Media Cybernetics, Silver Spring, Md.).

TABLE 16 Description of Healing Scores Healing Score Description High:Majority of collagen fibers have primary alignment, higher density ofcollagen fibers, some Sharpey's fibers present Medium: Some collagenfibers have primary alignment, medium density of collagen fibers, someSharpey's fibers present Low: Fewer collagen fibers have primaryalignment, lower density of collagen fibers with mediocre orientation,fewer or no Sharpey's Fibers presentResults(i)

In the first set of experiment (Collagen Matrix+0.15 mg/ml rhPDGF-BB or0.3 mg/ml rhPDGF-BB), all operated specimens, regardless of treatment,displayed some degree of tendon retraction after 12 weeks of healing. Onaverage, the infraspinatus tendon retracted 41.8±3.5 mm (mean±standarddeviation) from the bone trough for the 0.15 mg/ml rhPDGF-BB Dose group,and 45.2±8.9 mm for the 0.3 mg/ml rhPDGF-BB Dose group.

Histopathological scores averaged by treatment in the first set ofexperiment are presented in Table 17; graphical representation of theseresults is presented in FIG. 18. The sutures were observed to be intactwithin the bone tunnel for 4 of the 6 specimens after 12 weeks ofhealing, which indicates that the failure leading to the tendonretraction occurred at the suture-tendon interface and not thebone-suture interface.

Overall, regardless of treatment, reparative tissue between the humerusand native tendon end consisted of a fibrovascular tissue (highlyvascularized fibrous tissue) with active fibroplasia and moderatelydense, polarizable collagen fibers present. No differences in fibroblastdensity were observed between treatments. All specimens displayedprimary collagen fiber alignment parallel to that of the original tendonwith pockets of less organized fibers. Average collagen fiberorientation and fiber density was similar between treatments.

TABLE 17 Histopathological Scores Grouped by Treatment and AveragedBone-Tendon interface (Interdigitation/Sharpey's Fibers between tendonand Tendon End bone) Position 0 = 0% Tendon-Bone 1 = Tendon attachmentArea Integrated attached to 1 = 25% Tendon-Bone Collagen Fiber humerusInflammatory attachment Area Integrated Orientation Collagen 2 = TendonCells Vascularization 2 = 50% Tendon-Bone 0 = None Fiber Densityslightly retracted 0 = None 0 = None attachment Area Integrated 1 = Some1 = Low 3 = Tendon fully 1 = Some 1 = Some 3 = 75% Tendon-Bone 2 =Mostly 2 = Med Treatment retracted 2 = Many 2 = Abundant attachment AreaIntegrated 3 = Completely 3 = High 0.15 mg/ml rhPDGF-BB Average 3.0 0.82.0 1.6 1.7 2.5 StDev 0.0 0.2 0.0 0.5 0.6 0.0 0.3 mg/ml rhPDGF-BBAverage 3.0 0.9 2.0 1.4 1.8 2.1 StDev 0.0 0.3 0.0 1.5 0.2 0.3

At the regenerating insertion sites collagenous Sharpey's fibers of thereparative tendon tissue inserting and interdigitating either directlywith bone collagen or through a layer of fibrocartilage were observedfor all specimens. On average, interdigitation was observed overapproximately 30-40% of the total decorticated bone surface, and thisobservation was consistent across all treatment groups. The Sharpeyfibers observed in the operated specimens were more immature than theintact control; however, there was insertion and continuity of theseregenerative fibers with the collagen of the underlying bone. In a fewcases a portion of the original native tendon attachment site wasobserved in the operated shoulders.

Previous osteoclastic resorption of the bone surface was observed inmost specimens in the decorticated region of the original attachmentsite. It was recognized by the scalloped surface of the bone (Howship'slacunae) where osteoclasts were no longer present or a scallopedbasophilic reversal line where the surface had been covered by new bonetissue. Typically, the regions of previous resorption were covered witha layer of reactive woven bone with osteoblasts present. The reactivebone often had Sharpey fiber insertion. Resorption extent was variable.It was found over approximately 10-50% of the total bone surfaceregardless of treatment, and was typically covered by new woven bone.Islands of reactive woven bone and/or fibrocartilage were occasionallyobserved within the reparative tissue.

In all specimens, mild foreign body inflammation was observed within thehealing tissue and concentrated mainly near the suture material. In afew cases, small pockets of mononuclear inflammation were observedwithin the reparative tissue, possibly associated with vascularizationor focally damaged tissue. Inflammation was mainly mononuclear withmulti-nucleated giant cells. No neutrophils were typically observed.Abundant vascularization was observed in the healing tissue in allspecimens regardless of treatment. Angioblastic proliferation,indicating new vessel production, was also observed in a few specimensand was not correlated with a specific treatment. This proliferation wasmost probably due to the ongoing adaptational changes associated withthe healing process.

Fatty infiltration is known to be one consequence of rotator cuff tendontears, and has been shown to correlate with the degree of tendonretraction (Nakagaki et al, J. Clin Orth Rel Res (2008) and BjorkenheimJ. M., et al, Acta Orthop Scand. 60(4):461-3(1989). Fatty infiltrationwas observed in a few specimens in the peripheral tissue adjacent to themuscle. This was only observed in the center of the reparative tissue inone specimen (0.3 mg/ml rhPDGF-BB Dose).

(ii)

In the second set of experiment (Suture, Suture+collagen Matrix+AcetateBuffer, Suture+Collagen Matrix+rhPDGF-BB, or intact Control), theinfraspinatus tendon retracted 28.1±2.8 mm from the bone trough for theSuture Only group, 39.0±4.6 mm for the Suture+Collagen Matrix+AcetateBuffer group, and 40.9±8.3 mm for the Suture+Collagen Matrix+rhPDGF-BBgroup.

The sutures were intact within the bone tunnel for all specimens after12 weeks of healing which indicates that the failure leading to thetendon retraction occurred at the suture-tendon interface and not thebone-suture interface. Healing within and between treatments wasvariable. Healing for the Suture Only group was the most variable, withspecimens ranging from the best healing to the worst healing of thegroups. Suture+Collagen Matrix+Acetate Buffer specimens ranged ingrading from medium/high to medium/low healing, and Suture+CollagenMatrix+rhPDGF-BB specimens ranged from medium/low to low healing. Thetreatment itself was not visible in any of the stained histologicalslides. Healing scores for all specimens arranged best healing to worsthealing of the groups are shown in Table 18.

TABLE 18 Healing Scores for Individual Specimens specimen TreatmentHealing Score BS32 Suture Only High BS12 Suture + Collagen Matrix +Acetate Buffer Medium/High BS11 Suture Only Medium BS13 Suture +Collagen Matrix + Acetate Buffer Medium BS9 Suture + Collagen Matrix +rhPGDF-BB Medium/Low BS24 Suture + Collagen Matrix + Acetate BufferMedium/Low BS30 Suture + Collagen Matrix + rhPGDF-BB Medium/Low BS19Suture + Collagen Matrix + rhPGDF-BB Low BS6 Suture Only Low

Histopathological scores averaged by treatment are presented in Table 19and FIG. 19 Overall, regardless of treatment, reparative tissue betweenthe humerus and native tendon end consisted of a fibrovascular tissue(highly vascularized fibrous tissue) with active fibroplasia andpolarizable collagen fibers present. No differences in fibroblastdensity were observed between treatments. Some specimens had regions ofprimary collagen fiber alignment (with collagen alignment parallel tothat of the original tendon) and others did not; most specimens hadregions of both organized and unorganized collagen fiber alignment. Ingeneral, collagen alignment was better near the bone surface rather thannear the retracted tendon end.

TABLE 19 Histopathological Scores Grouped by Treatment and AveragedBone-Tendon Interface (Interdigitation/Sharpey's Fibers between tendonand bone, increments of 0.25) 0 = 0% Tendon-Bone attachment AreaIntegrated Tendon End Position 1 = 25% Tendon-Bone attachment 1 = Tendonattached to Area Integrated humerus Inflammatory 2 = 50% Tendon-Boneattachment 2 = Tendon slightly Cells Vascularization Area Integratedretracted 0 = None 0 = None 3 = 75% Tendon-Bone attachment 3 = Tendonfully 1 = Some 1 = Some Area Integrated Treatment retracted 2 = Many 2 =Abundant 4 = 100% Tendon-Bone attachment Suture Only Average 3 1 2 0.4StDev 0 0 0 0.2 Suture + Collagen Matrix + Average 3 1.2 2 0.3 AcetateBuffer StDev 0 0.2 0 0.2 Suture + Collagen Matrix + Average 3 1 2 0.1rhPGDF-BB StDev 0 0 0 0.1

In general, the collagenous Sharpey fibers of the tendon at itsinsertion and their interdigitation with bone collagen through a layerof fibrocartilage were observed, but in very small regions;Interdigitation was usually observed over less than 10% of the totalbone attachment surface. Small regions of Sharpey fiber insertion wereobserved in all three Suture Only (alone) specimens, in two of theSuture+Collagen Matrix+Acetate Buffer specimens, and in allSuture+Collagen Matrix+rhPDGF-BB specimens. In a few cases a portion ofthe original native tendon attachment site was observed in the operatedshoulders.

Osteoclastic resorption of the bone surface was observed in mostspecimens in the decorticated region of the original attachment site. Itwas recognized by the scalloped surface of the bone (Howship's lacunae)where osteoclasts were no longer present or the surface had been coveredby other tissue. Resorption occurred over approximately 10-20% of thetotal bone surface regardless of treatment. Reactive woven bone and/orfibrocartilage was observed within the reparative tissue and at thesurface of the bone in all three Suture Only specimens and oneSuture+Collagen Matrix+Acetate Buffer specimen.

In all specimens, mild foreign body inflammation was observed within thehealing tissue concentrated mainly near the suture material. In a fewcases, small pockets of mononuclear inflammation were observed withinthe reparative tissue, possibly associated with focally damaged tissue.Inflammation was mainly mononuclear with multi-nucleated giant cells.Generally, no neutrophils were observed. Abundant vascularization wasobserved in the healing tissue in all specimens regardless of treatment.Angioblast proliferation, indicating new vessel production, was alsoobserved in a few specimens regardless of treatment and probablyrepresented ongoing adaptational changes in the healing process.

CONCLUSION

Augmentation of a humural infraspinatus tendon reattachment with anrhPDGF-BB soaked collagen matrix did not prevent failure at thesuture-tissue interface. The tendon was retracted from the humerus andreplaced by reparative fibrovascular tissue in all specimens after 12weeks of healing, which suggests that retraction occurred within thefirst several weeks postoperatively.

Specimens exhibited varying degrees of new bone formation, inflammation,vascularity, and Sharpey's fiber's inserting the tendon to the bone atthe insertion site. No differences were noted in the suture only,suture+collagen, and suture+collagen+1.0 mg/mlrhPDGF-BB groups in theassessment of tendon retraction, inflammatory cells, vascularization, orSharpey's fibers. Histologic sections of the Suture+Collagen Matrix+0.15mg/ml rhPDGF-BB and Suture+Collagen Matrix+0.3 mg/ml rhPDGF-BB groupsdisplayed increased tendon repair and interdigitation of tendon collagenwith that of bone at the fibrocartilage interface. FIGS. 20A and 20B.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary. One of skill in the art will alsoappreciate that any methods and materials similar or equivalent to thosedescribed herein can also be used to practice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

What is claimed is:
 1. A method for attaching a ligament to a bone in anindividual comprising administering at an interface between the tendonand the bone or the ligament and the bone an effective amount of acomposition comprising: a solution of platelet-derived growth factor(PDGF) at a concentration of 0.1 to 2.0 mg/mL disposed in abiocompatible collagen matrix, wherein the biocompatible collagen matrixhas a porosity of at least 80%, and wherein at least about 50% of thePDGF is released within about 24 hours.
 2. The method of claim 1,wherein the biocompatible matrix has a porosity of at least about 90%.3. The method of claim 1, wherein the biocompatible matrix has aporosity of at least about 92%.
 4. The method of claim 1, wherein thebiocompatible matrix has a porosity of at least about 95%.
 5. The methodof claim 1, wherein the pores have an average area ranging from about2500 μm2 to about 20,000 μm2.
 6. The method of claim 1, wherein thepores have an average perimeter ranging from about 200 μm to about 600μm.
 7. The method of claim 1, wherein the pores have diameters rangingfrom about 1 μm to about 1 mm.
 8. The method of claim 7, wherein thepores have diameters at least about 5 μm.
 9. The method of claim 1,wherein the pores are interconnected pores.
 10. The method of claim 1,wherein at least about 60% of the PDGF is released within about 24hours.
 11. The method of claim 1, wherein at least about 70% of the PDGFis released within about 24 hours.
 12. The method of claim 1, wherein atleast about 80% of the PDGF is released within about 24 hours.
 13. Themethod of claim 1, wherein the biocompatible matrix is resorbed withinabout 21 days of in vivo administration.
 14. The method of claim 1,wherein the biocompatible matrix is resorbed within about 18 days of invivo administration.
 15. The method of claim 1, wherein thebiocompatible matrix is resorbed within about 15 days of in vivoadministration.
 16. The method of claim 1, wherein the collagen issoluble.
 17. The method of claim 1, wherein the collagen iscross-linked.
 18. The method of claim 1, wherein the biocompatiblematrix further comprises a glycosaminoglycan.
 19. The method of claim18, wherein the glycosaminoglycan is chondroitin sulfate.
 20. The methodof claim 1, wherein the composition is a sheet, pad, patch, gel orpaste.
 21. The method of claim 1, wherein the concentration of PDGF inthe solution is about 0.1 mg/ml to about 0.4 mg/ml.
 22. The method ofclaim 1, wherein the concentration of PDGF in the solution is about 0.9mg/ml to about 1.5 mg/ml.
 23. The method of claim 1, wherein cellsinfiltrate the composition within about 4 days after exposure to thecomposition.
 24. The method of claim 1, wherein the ligament is selectedfrom the group consisting of anterior cruciate ligament, lateralcollateral ligament, posterior cruciate ligament, medial collateralligament, cranial cruciate ligament, caudal cruciate ligament,cricothyroid ligament, periodontal ligament, suspensory ligament of thelens, anterior sacroiliac ligament, posterior sacroiliac ligament,sacrotuberous ligament, sacrospinous ligament, inferior pubic ligament,superior pubic ligament, suspensory ligament, palmar radiocarpalligament, dorsal radiocarpal ligament, ulnar collateral ligament, andradial collateral ligament.
 25. The method of claim 1, wherein theattachment is for anterior cruciate ligament reconstruction.
 26. Themethod of claim 1, wherein the collagen is cross-linked, and thebiocompatible matrix further comprises a glycosaminoglycan.
 27. Themethod of claim 26, wherein the glycosaminoglycan is chondroitinsulfate.
 28. The method of claim 26, wherein the weight/weight ratio ofcollagen to glycosaminoglycan is about 90:10.
 29. The method of claim26, wherein the weight/weight ratio of collagen to glycosaminoglycan isabout 92:8.
 30. The method of claim 26, wherein the weight/weight ratioof collagen to glycosaminoglycan is about 95:5.